Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

Science.gov (United States)

Parrell, Daniel; Zhang, Yang; Olenic, Sandra; Kroos, Lee

2017-10-01

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His 6 -MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His 6 -MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are

Activated H-Ras regulates hematopoietic cell survival by modulating Survivin

International Nuclear Information System (INIS)

Fukuda, Seiji; Pelus, Louis M.

2004-01-01

Survivin expression and Ras activation are regulated by hematopoietic growth factors. We investigated whether activated Ras could circumvent growth factor-regulated Survivin expression and if a Ras/Survivin axis mediates growth factor independent survival and proliferation in hematopoietic cells. Survivin expression is up-regulated by IL-3 in Ba/F3 and CD34 + cells and inhibited by the Ras inhibitor, farnesylthiosalicylic acid. Over-expression of constitutively activated H-Ras (CA-Ras) in Ba/F3 cells blocked down-modulation of Survivin expression, G 0 /G 1 arrest, and apoptosis induced by IL-3 withdrawal, while dominant-negative (DN) H-Ras down-regulated Survivin. Survivin disruption by DN T34A Survivin blocked CA-Ras-induced IL-3-independent cell survival and proliferation; however, it did not affect CA-Ras-mediated enhancement of S-phase, indicating that the anti-apoptotic activity of CA-Ras is Survivin dependent while its S-phase enhancing effect is not. These results indicate that CA-Ras modulates Survivin expression independent of hematopoietic growth factors and that a CA-Ras/Survivin axis regulates survival and proliferation of transformed hematopoietic cells

The radiosensitivity of human keratinocytes: influence of activated c-H-ras oncogene expression and tumorigenicity

International Nuclear Information System (INIS)

Mendonca, M.S.; Redpath, J.L.; Stanbridge, E.J.

1991-01-01

The authors investigated γ-ray sensitivity of several activated c-H-ras (EJ) containing clones established after transfection of the spontaneously immortalized non-tumorigenic human keratinocyte cell line HaCaT. The clones were grouped according to tumorigenic potential after subcutaneous injection into nude mice, and fell into three classes: Class I clones A-4 and I-6 are non-tumorigenic and express very low levels of c-H-ras mRNA and no mutated ras protein (p 21 ); Class II clones I-5 and I-7 grow to large (benign) epidermal cysts, express intermediate to high c-H-ras mRNA and variable levels of mutated ras p 21 protein with clone I-5 expressing little and clone I-7 expressing high levels of p 21 ; Class III clones II-3 and II-4 grow to solid squamous cell carcinomas, express high c-H-ras mRNA and high level of mutated p 21 ras protein similar to clone I-7. Comparison of single-hit multitarget or linear-quadratic survival curve parameters, and survival at 2Gy (S 2 ) indicate no general correlation with either activated c-H-ras expression level or tumorigenic potential, and increased radioresistance. (author)

Association between mutations of critical pathway genes and survival outcomes according to the tumor location in colorectal cancer.

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Lee, Dae-Won; Han, Sae-Won; Cha, Yongjun; Bae, Jeong Mo; Kim, Hwang-Phill; Lyu, Jaemyun; Han, Hyojun; Kim, Hyoki; Jang, Hoon; Bang, Duhee; Huh, Iksoo; Park, Taesung; Won, Jae-Kyung; Jeong, Seung-Yong; Park, Kyu Joo; Kang, Gyeong Hoon; Kim, Tae-You

2017-09-15

Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC. Targeted next-generation sequencing of 40 genes included in the 5 critical pathways of CRC (WNT, P53, RTK-RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase [PI3K], and transforming growth factor β [TGF-β]) was performed for 516 patients with stage III or high-risk stage II CRC treated with surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy. The associations between critical pathway mutations and relapse-free survival (RFS) and overall survival were analyzed. The associations were further analyzed according to the tumor location. The mutation rates for the WNT, P53, RTK-RAS, PI3K, and TGF-β pathways were 84.5%, 69.0%, 60.7%, 30.0%, and 28.9%, respectively. A mutation in the PI3K pathway was associated with longer RFS (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36-0.99), whereas a mutation in the RTK-RAS pathway was associated with shorter RFS (adjusted HR, 1.60; 95% CI, 1.01-2.52). Proximal tumors showed a higher mutation rate than distal tumors, and the mutation profile was different according to the tumor location. The mutation rates of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and B-Raf proto-oncogene serine/threonine kinase (BRAF) were higher in proximal tumors, and the mutation rates of adenomatous polyposis coli (APC), tumor protein 53 (TP53), and neuroblastoma RAS viral oncogene homolog (NRAS) were higher in distal tumors. The better RFS with the PI3K pathway mutation was significant only for proximal tumors, and the worse RFS with the RTK-RAS pathway mutation was significant only for distal tumors. A PI3K pathway mutation was associated with better RFS for CRC patients treated with adjuvant chemotherapy, and an RTK-RAS

Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

NARCIS (Netherlands)

Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

Evidence implicating the Ras pathway in multiple CD28 costimulatory functions in CD4+ T cells.

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Sujit V Janardhan

Full Text Available CD28 costimulation is a critical event in the full activation of CD4(+ T cells that augments cytokine gene transcription, promotes cytokine mRNA stability, prevents induction of anergy, increases cellular metabolism, and increases cell survival. However, despite extensive biochemical analysis of the signaling events downstream of CD28, molecular pathways sufficient to functionally replace the diverse aspects of CD28-mediated costimulation in normal T cells have not been identified. Ras/MAPK signaling is a critical pathway downstream of T cell receptor stimulation, but its role in CD28-mediated costimulation has been controversial. We observed that physiologic CD28 costimulation caused a relocalization of the RasGEF RasGRP to the T cell-APC interface by confocal microscopy. In whole cell biochemical analysis, CD28 cross-linking with either anti-CD28 antibody or B7.1-Ig augmented TCR-induced Ras activation. To determine whether Ras signaling was sufficient to functionally mimic CD28 costimulation, we utilized an adenoviral vector encoding constitutively active H-Ras (61L to transduce normal, Coxsackie-Adenovirus Receptor (CAR transgenic CD4(+ T cells. Like costimulation via CD28, active Ras induced AKT, JNK and ERK phosphorylation. In addition, constitutive Ras signaling mimicked the ability of CD28 to costimulate IL-2 protein secretion, prevent anergy induction, increase glucose uptake, and promote cell survival. Importantly, we also found that active Ras mimicked the mechanism by which CD28 costimulates IL-2 production: by increasing IL-2 gene transcription, and promoting IL-2 mRNA stability. Finally, active Ras was able to induce IL-2 production when combined with ionomycin stimulation in a MEK-1-dependent fashion. Our results are consistent with a central role for Ras signaling in CD28-mediated costimulation.

Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

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The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.

Science.gov (United States)

Tartaglia, Marco; Gelb, Bruce D

2010-12-01

RAS GTPases control a major signaling network implicated in several cellular functions, including cell fate determination, proliferation, survival, differentiation, migration, and senescence. Within this network, signal flow through the RAF-MEK-ERK pathway-the first identified mitogen-associated protein kinase (MAPK) cascade-mediates early and late developmental processes controlling morphology determination, organogenesis, synaptic plasticity, and growth. Signaling through the RAS-MAPK cascade is tightly controlled; and its enhanced activation represents a well-known event in oncogenesis. Unexpectedly, in the past few years, inherited dysregulation of this pathway has been recognized as the cause underlying a group of clinically related disorders sharing facial dysmorphism, cardiac defects, reduced postnatal growth, ectodermal anomalies, variable cognitive deficits, and susceptibility to certain malignancies as major features. These disorders are caused by heterozygosity for mutations in genes encoding RAS proteins, regulators of RAS function, modulators of RAS interaction with effectors, or downstream signal transducers. Here, we provide an overview of the phenotypic spectrum associated with germline mutations perturbing RAS-MAPK signaling, the unpredicted molecular mechanisms converging toward the dysregulation of this signaling cascade, and major genotype-phenotype correlations. © 2010 New York Academy of Sciences.

Evaluation of K-ras and p53 expression in pancreatic adenocarcinoma using the cancer genome atlas.

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Liming Lu

Full Text Available Genetic alterations in K-ras and p53 are thought to be critical in pancreatic cancer development and progression. However, K-ras and p53 expression in pancreatic adenocarcinoma have not been systematically examined in The Cancer Genome Atlas (TCGA Data Portal. Information regarding K-ras and p53 alterations, mRNA expression data, and protein/protein phosphorylation abundance was retrieved from The Cancer Genome Atlas (TCGA databases, and analyses were performed by the cBioPortal for Cancer Genomics. The mutual exclusivity analysis showed that events in K-ras and p53 were likely to co-occur in pancreatic adenocarcinoma (Log odds ratio = 1.599, P = 0.006. The graphical summary of the mutations showed that there were hotspots for protein activation. In the network analysis, no solid association between K-ras and p53 was observed in pancreatic adenocarcinoma. In the survival analysis, neither K-ras nor p53 were associated with both survival events. As in the data mining study in the TCGA databases, our study provides a new perspective to understand the genetic features of K-ras and p53 in pancreatic adenocarcinoma.

Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus

DEFF Research Database (Denmark)

Willumsen, B M; Norris, K; Papageorge, A G

1984-01-01

localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein...... not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue...

EGFR immunoexpression, RAS immunoexpression and their effects on survival in lung adenocarcinoma cases.

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Gundogdu, Ahmet Gokhan; Onder, Sevgen; Firat, Pinar; Dogan, Riza

2014-06-01

The impacts of epidermal growth factor receptor (EGFR) immunoexpression and RAS immunoexpression on the survival and prognosis of lung adenocarcinoma patients are debated in the literature. Twenty-six patients, who underwent pulmonary resections between 2002 and 2007 in our clinic, and whose pathologic examinations yielded adenocarcinoma, were included in the study. EGFR and RAS expression levels were examined by immunohistochemical methods. The results were compared with the survival, stage of the disease, nodal involvement, lymphovascular invasion, and pleural invasion. Nonparametric bivariate analyses were used for statistical analyses. A significant link between EGFR immunoexpression and survival has been identified while RAS immunoexpression and survival have been proven to be irrelevant. Neither EGFR, nor RAS has displayed a significant link with the stage of the disease, nodal involvement, lymphovascular invasion, or pleural invasion. Positive EGFR immunoexpression affects survival negatively, while RAS immunoexpression has no effect on survival in lung adenocarcinoma patients.

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

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Martín-Sánchez, Paloma; Luengo, Alicia; Griera, Mercedes; Orea, María Jesús; López-Olañeta, Marina; Chiloeches, Antonio; Lara-Pezzi, Enrique; de Frutos, Sergio; Rodríguez-Puyol, Manuel; Calleros, Laura; Rodríguez-Puyol, Diego

2018-02-01

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras -/- ) and control wild-type (H -ras +/+ ) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iβ protein expression in H -ras -/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras -/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3β-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iβ overexpression in H -ras -/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iβ overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

Atorvastatin inhibits insulin synthesis by inhibiting the Ras/Raf/ERK/CREB pathway in INS-1 cells

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Sun, Hongxi; Li, Yu; Sun, Bei; Hou, Ningning; Yang, Juhong; Zheng, Miaoyan; Xu, Jie; Wang, Jingyu; Zhang, Yi; Zeng, Xianwei; Shan, Chunyan; Chang, Bai; Chen, Liming; Chang, Baocheng

2016-01-01

Abstract Backround: Type 2 diabetes has become a global epidemic disease. Atorvastatin has become a cornerstone in the prevention and treatment of atherosclerosis. However, increasing evidence showed that statins can dose-dependently increase the risk of diabetes mellitus. The mechanism is not clear. Objective: The Ras complex pathway (Ras/Raf/extracellular signal-regulated kinase [ERK]/cAMP response element-binding protein [CREB]) is the major pathway that regulates the gene transcription. Except for the inhibition of cholesterol synthesis by inhibiting the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-COA) reductase, statins can also downregulate the phosphorylation of a series of downstream substrates including the key proteins of the Ras complex pathway, therefore may inhibit the insulin syntheses in pancreatic beta cells. In our study, we investigated the inhibitory effect and the underlying mechanism of atorvastatin on insulin synthesis in rat islets. Methods: Islets were isolated from Wistar rats and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. The insulin content in the medium was measured by radioimmunoassay before and after the treatment of 50 μM atorvastatin. Effect of atorvastatin on the expression of insulin message Ribonucleic acid (mRNA) in pancreatic islet beta cells was also detected using quantitative real-time polymerase chain reaction. Western blotting was used to explore the possible role of the Ras complex pathway (Ras/Raf/ERK/CREB) in atorvastatin-inhibited insulin synthesis. The effects of atorvastatin on the binding of nuclear transcription factor p-CREB with CRE in INS-1 cells were examined via chromatin immunoprecipitation assay. Results: Compared with the control group, the insulin level decreased by 27.1% at 24 hours after atorvastatin treatment. Atorvastatin inhibited insulin synthesis by decreasing insulin mRNA expression of pancreatic islet beta cells. The activities of Ras, Raf-1, and p-CREB in the Ras complex

Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

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Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

2017-09-16

Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

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Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

2009-10-20

Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

Interaction between Wnt/β-catenin and RAS-ERK pathways and an anti-cancer strategy via degradations of β-catenin and RAS by targeting the Wnt/β-catenin pathway.

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Jeong, Woo-Jeong; Ro, Eun Ji; Choi, Kang-Yell

2018-01-01

Aberrant activation of the Wnt/β-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as adenomatous polyposis coli ( APC ) and KRAS are frequently mutated in human CRC, and involved in the initiation and progression of the tumorigenesis, respectively. Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/β-catenin and RAS-ERK pathways is a stabilization of both β-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased β-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both β-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/β-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/β-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both β-catenin and RAS via targeting Wnt/β-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.

Ras and relatives--job sharing and networking keep an old family together.

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Ehrhardt, Annette; Ehrhardt, Götz R A; Guo, Xuecui; Schrader, John W

2002-10-01

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

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Clara L Oeste

2011-01-01

Full Text Available Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184 in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14-PGJ(2 (15d-PGJ(2 and Δ(12-PGJ(2 selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO, a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2. Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

Which downstream signal transduction pathway(s) of H-ras are necessary for the cellular response(s) to ionizing radiation? (Results of an astro research fellowship year)

International Nuclear Information System (INIS)

Rudoltz, Marc S.; Muschel, Ruth J.; McKenna, W. Gillies

1996-01-01

Purpose/Background: The H-ras oncogene encodes a protein which is an essential component of multiple downstream effector pathways required for induction of proliferation and differentiation. Ras plays a role in the control some of these signal transduction pathways, such as the MAP kinase pathway which controls gene expression and the Rac-Rho pathway which controls cell morphology. Previous work from our laboratory has associated H-ras expression with radiation resistance, a prolonged delay in G2 following exposure to ionizing radiation, and suppression of radiation-induced apoptosis. In addition, H-ras cooperates with myc in transformation. Recent work by White et al. (Cell 80:533-541, 1995) and Joneson et al. (Science 271: 810-812, 1996) describes three mutations in H-ras which were engineered to eliminate different downstream signal transduction pathways of H-ras. T35S contains a serine in place of threonine at amino acid 35 and is defective for ras-induced cytoskeletal changes and initiation of DNA synthesis. E37G contains a glutamic acid in place of glycine at amino acid 37 which eliminates interaction of H-ras with a GDP/GTP exchange factor. C40 contains a substitution of cysteine for tyrosine at amino acid 40 and is defective for H-ras induction of the MAP kinase pathway. We propose that by expressing these mutant H-ras proteins in immortalized cells the downstream pathways of H-ras which regulate the cellular response(s) to ionizing radiation may be determined. Materials and Methods: pHP-5 plasmids encoding these H-ras mutant genes (see White et al.) were transfected by calcium phosphate precipitation into MR4 cells, rat embryo fibroblasts immortalized by expression of v-myc. In this vector, the cDNA for H-ras is placed under the control of a CMV constitutive promoter, and selection is provided by hygromycin. The transfections performed were as follows: V12Ras (no mutation), T35S, E37G, C40, T35S + E37G, and T35S + C40. Twenty four hours after transfection

An orthosteric inhibitor of the RAS-SOS interaction.

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Nickerson, Seth; Joy, Stephen T; Arora, Paramjit S; Bar-Sagi, Dafna

2013-01-01

Rat sarcoma (RAS) proteins are signaling nodes that transduce extracellular cues into precise alterations in cellular physiology by engaging effector pathways. RAS signaling thus regulates diverse cell processes including proliferation, migration, differentiation, and survival. Owing to this central role in governing mitogenic signals, RAS pathway components are often dysregulated in human diseases. Targeted therapy of RAS pathways has generally not been successful, largely because of the robust biochemistry of the targets and their multifaceted network of molecular regulators. The rate-limiting step of RAS activation is Son of Sevenless (SOS)-mediated nucleotide exchange involving a single evolutionarily conserved catalytic helix from SOS. Structure function data of this mechanism provided a strong platform to design an SOS-derived, helically constrained peptide mimic as an inhibitor of the RAS-SOS interaction. In this chapter, we review RAS-SOS signaling dynamics and present evidence supporting the novel paradigm of inhibiting their interaction as a therapeutic strategy. We then describe a method of generating helically constrained peptide mimics of protein surfaces, which we have employed to inhibit the RAS-SOS active site interaction. The biochemical and functional properties of this SOS mimic support the premise that inhibition of RAS-nucleotide exchange can effectively block RAS activation and downstream signaling. © 2013 Elsevier Inc. All rights reserved.

Machine Learning Detects Pan-cancer Ras Pathway Activation in The Cancer Genome Atlas

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Gregory P. Way

2018-04-01

Full Text Available Summary: Precision oncology uses genomic evidence to match patients with treatment but often fails to identify all patients who may respond. The transcriptome of these “hidden responders” may reveal responsive molecular states. We describe and evaluate a machine-learning approach to classify aberrant pathway activity in tumors, which may aid in hidden responder identification. The algorithm integrates RNA-seq, copy number, and mutations from 33 different cancer types across The Cancer Genome Atlas (TCGA PanCanAtlas project to predict aberrant molecular states in tumors. Applied to the Ras pathway, the method detects Ras activation across cancer types and identifies phenocopying variants. The model, trained on human tumors, can predict response to MEK inhibitors in wild-type Ras cell lines. We also present data that suggest that multiple hits in the Ras pathway confer increased Ras activity. The transcriptome is underused in precision oncology and, combined with machine learning, can aid in the identification of hidden responders. : Way et al. develop a machine-learning approach using PanCanAtlas data to detect Ras activation in cancer. Integrating mutation, copy number, and expression data, the authors show that their method detects Ras-activating variants in tumors and sensitivity to MEK inhibitors in cell lines. Keywords: Gene expression, machine learning, Ras, NF1, KRAS, NRAS, HRAS, pan-cancer, TCGA, drug sensitivity

Ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

International Nuclear Information System (INIS)

Comerford, J.G.; Gibson, J.R.; Dawson, A.P.; Gibson, I.

1989-01-01

The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma- 35 S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma- 35 S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines

Utilizing ras signaling pathway to direct selective replication of herpes simplex virus-1.

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Weihong Pan

Full Text Available Re-engineering the tropism of viruses is an attractive translational strategy for targeting cancer cells. The Ras signal transduction pathway is a central hub for a variety of pro-oncogenic events with a fundamental role in normal and neoplastic physiology. In this work we were interested in linking Ras activation to HSV-1 replication in a direct manner in order to generate a novel oncolytic herpes virus which can target cancer cells. To establish such link, we developed a mutant HSV-1 in which the expression of ICP4 (infected cell protein-4, a viral protein necessary for replication is controlled by activation of ELK, a transcription factor down-stream of the Ras pathway and mainly activated by ERK (extracellular signal-regulated kinase, an important Ras effector pathway. This mutant HSV-1 was named as Signal-Smart 1 (SS1. A series of prostate cells were infected with the SS1 virus. Cells with elevated levels of ELK activation were preferentially infected by the SS1 virus, as demonstrated by increased levels of viral progeny, herpetic glycoprotein C and overall SS1 viral protein production. Upon exposure to SS1, the proliferation, invasiveness and colony formation capabilities of prostate cancer cells with increased ELK activation were significantly decreased (p<0.05, while the rate of apoptosis/necrosis in these cells was increased. Additionally, high Ras signaling cells infected with SS1 showed a prominent arrest in the G1 phase of the cell cycle as compared to cells exposed to parental HSV-1. The results of this study reveal the potential for re-modeling the host-herpes interaction to specifically interfere with the life of cancer cells with increased Ras signaling. SS1 also serves as a "prototype" for development of a family of signal-smart viruses which can target cancer cells on the basis of their signaling portfolio.

EMT-induced stemness and tumorigenicity are fueled by the EGFR/Ras pathway.

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Dominic Chih-Cheng Voon

Full Text Available Recent studies have revealed that differentiated epithelial cells would acquire stem cell-like and tumorigenic properties following an Epithelial-Mesenchymal Transition (EMT. However, the signaling pathways that participate in this novel mechanism of tumorigenesis have not been fully characterized. In Runx3 (-/- p53 (-/- murine gastric epithelial (GIF-14 cells, EMT-induced plasticity is reflected in the expression of the embryonal proto-oncogene Hmga2 and Lgr5, an exclusive gastrointestinal stem cell marker. Here, we report the concurrent activation of an EGFR/Ras gene expression signature during TGF-β1-induced EMT in GIF-14 cells. Amongst the altered genes was the induction of Egfr, which corresponded with a delayed sensitization to EGF treatment in GIF-14. Co-treatment with TGF-β1 and EGF or the expression of exogenous KRas led to increased Hmga2 or Lgr5 expression, sphere initiation and colony formation in soft agar assay. Interestingly, the gain in cellular plasticity/tumorigenicity was not accompanied by increased EMT. This uncoupling of EMT and the induction of plasticity reveals an involvement of distinct signaling cues, whereby the EGFR/Ras pathway specifically promotes stemness and tumorigenicity in EMT-altered GIF-14 cells. These data show that the EGFR/Ras pathway requisite for the sustenance of gastric stem cells in vivo and in vitro is involved in the genesis and promotion of EMT-induced tumor-initiating cells.

Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

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Puil, L; Liu, J; Gish, G; Mbamalu, G; Bowtell, D; Pelicci, P G; Arlinghaus, R; Pawson, T

1994-01-01

The cytosolic 185 and 210 kDa Bcr-Abl protein tyrosine kinases play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph+ ALL). p185 and p210 Bcr-Abl contain identical abl-encoded sequences juxtaposed to a variable number of bcr-derived amino acids. As the mitogenic and transforming activities of tyrosine kinases involve stimulation of the Ras pathway, we analyzed Bcr-Abl oncoproteins for interacti...

The regulation of ras-raf signaling pathway on G1 phase of the irradiated cells

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Guo Dehuang; Dong Bo; Liu Nongle; Wen Gengyun; Luo Qingliang; Mao Bingzhi

2000-01-01

Objective: To investigate the way of ras-raf signaling pathway which regulate the G 1 phase in irradiated KG-1 cells. Methods: Blocked the GM-CSF signaling pathway by transfected DN-ras and then momentary transfected cyclin D1 into irradiated KG-1 cells, the effects of cyclin D1 on G 1 phase was examined. Results: The irradiated KG-1 cells transfected DN-ras can't recover form G 1 phase arrest even though the GM-CSF was given,momentary transfected cyclin D1 promote the irradiated KG-1 cells from G 1 arrest. Conclusion: Activation of ras-raf signaling pathway regulate the cell cycle of the irradiated KG-1 cells through promotion the expression of the cyclin D1

Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

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Tseng, Ya-Shih; Lee, Jenq-Chang; Huang, Chi-Ying F; Liu, Hsiao-Sheng

2009-01-01

Overexpression of Aurora-A and mutant Ras (Ras V12 ) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear. Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-ras mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either Ras V12 and wild-type Aurora-A (designated WT) or Ras V12 and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved. Overexpression of wild-type Aurora-A and mutation of Ras V12 were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the Ras V12 transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the Ras V12 transformants. Wild-type-Aurora-A enhances focus formation and aggregation of the Ras V12 transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

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Lechuga, Carmen G; Simón-Carrasco, Lucía; Jacob, Harrys K C; Drosten, Matthias

2017-01-01

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Ras lox (H-Ras -/- , N-Ras -/- , K-Ras lox/lox , RERT ert/ert ) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

Opposite effects of Ha-Ras and Ki-Ras on radiation-induced apoptosis via differential activation of PI3K/Akt and Rac/p38 mitogen-activated protein kinase signaling

International Nuclear Information System (INIS)

Choi, J.-A.; Kang, C.-M.; Lee, Y.-S.; Lee, S.-J.; Bae, S.-W.; Cho, C.-K.

2003-01-01

It has been well known that Ras signaling is involved in various cellular processes, including proliferation, differentiation, and apoptosis. However, distinct cellular functions of Ras isozymes are not fully understood. Here we show the opposing roles of Ha-Ras and Ki-Ras genes in the modulation of cell sensitivity to ionizing radiation. Overexpression of active isoform of Ha-Ras (12V-Ha- Ras) in Rat2 cells increases resistance to the ionizing radiation. Constitutive activation of phosphoinositide-3-kinase (PI3K) and Akt is detected specifically in 12V-Ha-Ras-overexpressing cells. The specific PI3K inhibitor LY294002 inhibits PI3K/Akt signaling and potentiates the radiation-induced apoptosis, suggesting that activation of PI3K/Akt signaling pathway is involved in the increased radio-resistance in cells overexpressing 12V-Ha-Ras. Overexpression of activated Ki-Ras (12V-Ki-Ras), on the other hand, markedly increases radiation sensitivity. The p38 mitogen-activated protein (MAP) kinase activity is selectively enhanced by ionizing radiation in cells overexpressing 12V-Ki-Ras. The specific p38 MAP kinase inhibitor, PD169316, or dominant-negative p38 MAP kinase decreases radiation-induced cell death. We further show that the mechanism that underlies potentiation of cell death in cells overexpressing 12V-Ki-Ras involves Bax translocation to the mitochondrial membrane. Elevated Bax translocation following ionizing irradiation in 12V-Ki-Ras-overexpressing cells is completely inhibited by PD169316 or dominant-negative p38 MAP kinase. In addition, introduction of cells with RacN17, a dominant negative mutant of Rac, resulted in a marked inhibition of radiation-induced Bax translocation and apoptotic cell death as well as p38 MAP kinase activation. Taken together, these findings explain the opposite effects of Ha-Ras and Ki-Ras on modulation of radio-sensitivity, and suggest that differential activation of PI3K/Akt and Rac/p38 MAP kinase signaling by Ha-Ras and Ki-Ras may

PI3K: A Crucial Piece in the RAS Signaling Puzzle.

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Krygowska, Agata Adelajda; Castellano, Esther

2018-06-01

RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, is a putative tumor suppressor.

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Kanno, Emiri; Kawasaki, Osamu; Takahashi, Kazuya; Takano, Kazunori; Endo, Takeshi

2018-01-01

Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf-MEK-ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

RAS signalling in energy metabolism and rare human diseases.

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Dard, L; Bellance, N; Lacombe, D; Rossignol, R

2018-05-08

The RAS pathway is a highly conserved cascade of protein-protein interactions and phosphorylation that is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Recent findings indicate that the RAS pathway plays a role in the regulation of energy metabolism via the control of mitochondrial form and function but little is known on the participation of this effect in RAS-related rare human genetic diseases. Germline mutations that hyperactivate the RAS pathway have been discovered and linked to human developmental disorders that are known as RASopathies. Individuals with RASopathies, which are estimated to affect approximately 1/1000 human birth, share many overlapping characteristics, including cardiac malformations, short stature, neurocognitive impairment, craniofacial dysmorphy, cutaneous, musculoskeletal, and ocular abnormalities, hypotonia and a predisposition to developing cancer. Since the identification of the first RASopathy, type 1 neurofibromatosis (NF1), which is caused by the inactivation of neurofibromin 1, several other syndromes have been associated with mutations in the core components of the RAS-MAPK pathway. These syndromes include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), which was formerly called LEOPARD syndrome, Costello syndrome (CS), cardio-facio-cutaneous syndrome (CFC), Legius syndrome (LS) and capillary malformation-arteriovenous malformation syndrome (CM-AVM). Here, we review current knowledge about the bioenergetics of the RASopathies and discuss the molecular control of energy homeostasis and mitochondrial physiology by the RAS pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1.

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Vercoulen, Yvonne; Kondo, Yasushi; Iwig, Jeffrey S; Janssen, Axel B; White, Katharine A; Amini, Mojtaba; Barber, Diane L; Kuriyan, John; Roose, Jeroen P

2017-09-27

RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

Parallel imaging of Drosophila embryos for quantitative analysis of genetic perturbations of the Ras pathway

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Yogesh Goyal

2017-07-01

Full Text Available The Ras pathway patterns the poles of the Drosophila embryo by downregulating the levels and activity of a DNA-binding transcriptional repressor Capicua (Cic. We demonstrate that the spatiotemporal pattern of Cic during this signaling event can be harnessed for functional studies of mutations in the Ras pathway in human diseases. Our approach relies on a new microfluidic device that enables parallel imaging of Cic dynamics in dozens of live embryos. We found that although the pattern of Cic in early embryos is complex, it can be accurately approximated by a product of one spatial profile and one time-dependent amplitude. Analysis of these functions of space and time alone reveals the differential effects of mutations within the Ras pathway. Given the highly conserved nature of Ras-dependent control of Cic, our approach provides new opportunities for functional analysis of multiple sequence variants from developmental abnormalities and cancers.

S1P/S1PR3 signaling mediated proliferation of pericytes via Ras/pERK pathway and CAY10444 had beneficial effects on spinal cord injury.

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Tang, Hai-Bin; Jiang, Xiao-Jian; Wang, Chen; Liu, Shi-Chang

2018-04-15

Pericytes have long been regarded merely to maintain structural and functional integrity of blood-brain barrier (BBB). Nevertheless, it has also been identified as a component of scar-forming stromal cells after spinal cord injury (SCI). In process of enlargement of spinal cavity after SCI, the number of pericytes increased and outnumbered astrocytes. However, the mechanism of proliferation of pericytes remains unclear. Sphingosine-1-phosphate (S1P) has been reported to play important roles in the formation of glia scar, but previous studies had paid more attention to the astrocytes. The present study aimed to observe the effects of S1P and S1P receptors (S1PRs) on proliferation of pericytes and investigate the underlying mechanism. By double immunostaining, we found that the number of PDGFRβ-positive pericytes was gradually increased and sealed the cavity, which surrounded by reactive astrocytes. Moreover, the subtype of S1PR3 was found to be induced by SCI and mainly expressed on pericytes. Further, by use of CAY10444, an inhibitor of S1PR3, we showed that S1P/S1PR3 mediated the proliferation of pericytes through Ras/pERK pathway. Moreover, CAY10444 was found to have the effects of enhancing neuronal survival, alleviating glial scar formation, and improving locomotion recovery after SCI. The results suggested that S1P/S1PR3 might be a promising target for clinical therapy for SCI. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

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Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

2008-03-01

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

International Nuclear Information System (INIS)

Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

2012-01-01

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ∼ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ∼ 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

Energy Technology Data Exchange (ETDEWEB)

Naumov, Inna [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Kazanov, Dina [Integrated Cancer Prevention Center, Tel Aviv (Israel); Lisiansky, Victoria [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Starr, Alex [Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Aroch, Ilan; Shapira, Shiran; Kraus, Sarah [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Arber, Nadir, E-mail: narber@post.tau.ac.il [Integrated Cancer Prevention Center, Tel Aviv (Israel); Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel)

2012-01-15

Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

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Mark Merchant

Full Text Available Mitogen-activated protein kinase (MAPK pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and

Gain-of-function mutations in RIT1 cause Noonan syndrome, a RAS/MAPK pathway syndrome.

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Aoki, Yoko; Niihori, Tetsuya; Banjo, Toshihiro; Okamoto, Nobuhiko; Mizuno, Seiji; Kurosawa, Kenji; Ogata, Tsutomu; Takada, Fumio; Yano, Michihiro; Ando, Toru; Hoshika, Tadataka; Barnett, Christopher; Ohashi, Hirofumi; Kawame, Hiroshi; Hasegawa, Tomonobu; Okutani, Takahiro; Nagashima, Tatsuo; Hasegawa, Satoshi; Funayama, Ryo; Nagashima, Takeshi; Nakayama, Keiko; Inoue, Shin-Ichi; Watanabe, Yusuke; Ogura, Toshihiko; Matsubara, Yoichi

2013-07-11

RAS GTPases mediate a wide variety of cellular functions, including cell proliferation, survival, and differentiation. Recent studies have revealed that germline mutations and mosaicism for classical RAS mutations, including those in HRAS, KRAS, and NRAS, cause a wide spectrum of genetic disorders. These include Noonan syndrome and related disorders (RAS/mitogen-activated protein kinase [RAS/MAPK] pathway syndromes, or RASopathies), nevus sebaceous, and Schimmelpenning syndrome. In the present study, we identified a total of nine missense, nonsynonymous mutations in RIT1, encoding a member of the RAS subfamily, in 17 of 180 individuals (9%) with Noonan syndrome or a related condition but with no detectable mutations in known Noonan-related genes. Clinical manifestations in the RIT1-mutation-positive individuals are consistent with those of Noonan syndrome, which is characterized by distinctive facial features, short stature, and congenital heart defects. Seventy percent of mutation-positive individuals presented with hypertrophic cardiomyopathy; this frequency is high relative to the overall 20% incidence in individuals with Noonan syndrome. Luciferase assays in NIH 3T3 cells showed that five RIT1 alterations identified in children with Noonan syndrome enhanced ELK1 transactivation. The introduction of mRNAs of mutant RIT1 into 1-cell-stage zebrafish embryos was found to result in a significant increase of embryos with craniofacial abnormalities, incomplete looping, a hypoplastic chamber in the heart, and an elongated yolk sac. These results demonstrate that gain-of-function mutations in RIT1 cause Noonan syndrome and show a similar biological effect to mutations in other RASopathy-related genes. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The Prognostic Impact of K-RAS Mutations in Adult Acute Myeloid Leukemia Patients Treated with High Dose Cytarabine

International Nuclear Information System (INIS)

Ahmad, E.I.; Gawish, H.H.; Al-Azizi, N.M.A.; El-Hefni, A.M.

2009-01-01

Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML). However there is still controversy about its clinical relevance on the treatment outcome of this leukemia. Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C) used in post induction consolidation chemotherapy in adult AML patients. Patients and Methods: The study comprised 71de novo AML patients with a male: Female ratio of 1.4: 1; their ages ranged from 21-59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G banding and K-RAS mutation detection using realtime PCR. The patients were randomized into 2 groups (gps) according to the ara-C dose used in consolidation treatment, HDAC gp receiving 400 mg ara-C and LDAC gp receiving 100 mg ara-C. They were followed over a period of 5 years. Results: Mutations in the K-RAS gene (mutRAS) were detected in 23 patients (32%) with the remaining 48 patients (68%) having wild type RAS (wtRAS). Blast cell percentage was significantly lower in mutRAS compared to wtRAS patients (p=<0.001). The M4 subtype of AML and cases with Inv 16 showed significantly higher frequencies in mutRAS compared to wtRAS patients, (p=0.015, 0.003, respectively). The patients were followed up for a median of 43 months (range 11-57 months). There was no significant difference in overall survival (OS) between mutRAS and wtRAS patients (p=0.326). Within the mutRAS patients treated with HDAC, cumulative OS was significantly higher than those treated with LDAC (p=0.001). This was not the case in the wtRAS group (p=0.285). There was no significant difference in disease The Prognostic Impact of K-RAS Mutations in Adult Acute Myeloid Leukemia Patients Treated with High Dose

Biological aspects and tumorigenic activity of the Ras proto-oncogenic family Aspectos biológicos e atividade tumorigênica da família proto-oncogênica Ras

Directory of Open Access Journals (Sweden)

Juliano André Boquett

2010-12-01

Full Text Available Proto-oncogenes play an important role in the regulation of the cellular cycle, being critical to the tumorigenesis. In this category we can find the RAS family. Due to the high transformation potential of these genes, this family is the best described and most studied one. It is formed by the H-, K- and the N-RAS genes, that codify highly related proteins expressed in several types of cells, denominated p21.These proteins act in the sign transduction from the membrane to the nucleus, as well as in the control of proliferation, differentiation and cellular death, and they are regulated by the interaction with GDP (inactive and GTP (active. These proteins show variation in only 10 - 15% of the primary structure, in the C-terminal portion denominated hyper-variant region. When in the oncogenic form, the p21 proteins remain active, providing continuous stimuli to the cellular proliferation. Among the RAS genes, K-RAS ones have been the most studied for presenting more frequent mutations and for being present in more aggressive tumors, implying the patients’ shorter survival time. Due to these facts and relative bibliography lack in the Portuguese language on this family, we presented in this work a systematized and updated review on the RAS genes. Os proto-oncogenes desempenham importante papel na regulação do ciclo celular, e são críticos à tumorigênese. Nessa categoria se encontra a família RAS, que, devido ao elevado potencial transformante dos genes que a compõem, é uma das mais bem descritas e estudadas. É formada pelos genes H-, K- e N-RAS, que codificam proteínas altamente relacionadas expressas em vários tipos de células, denominadas p21. Estas atuam na transdução de sinal da membrana ao núcleo, estão envolvidas no controle da proliferação, diferenciação e morte celular e são reguladas pela interação com GDP (inativa e GTP (ativa. As proteínas p21 diferem em apenas 10-15% da sua estrutura primária, na porção C

The effect of aquaporin 5 overexpression on the Ras signaling pathway

International Nuclear Information System (INIS)

Woo, Janghee; Lee, Juna; Kim, Myoung Sook; Jang, Se Jin; Sidransky, David; Moon, Chulso

2008-01-01

Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells

Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

DEFF Research Database (Denmark)

Adari, H; Lowy, D R; Willumsen, B M

1988-01-01

A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as w...

Adaptation of a RAS pathway activation signature from FF to FFPE tissues in colorectal cancer

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Bernard Omolo

2016-10-01

Full Text Available Abstract Background The KRAS gene is mutated in about 40 % of colorectal cancer (CRC cases, which has been clinically validated as a predictive mutational marker of intrinsic resistance to anti-EGFR inhibitor (EGFRi therapy. Since nearly 60 % of patients with a wild type KRAS fail to respond to EGFRi combination therapies, there is a need to develop more reliable molecular signatures to better predict response. Here we address the challenge of adapting a gene expression signature predictive of RAS pathway activation, created using fresh frozen (FF tissues, for use with more widely available formalin fixed paraffin-embedded (FFPE tissues. Methods In this study, we evaluated the translation of an 18-gene RAS pathway signature score from FF to FFPE in 54 CRC cases, using a head-to-head comparison of five technology platforms. FFPE-based technologies included the Affymetrix GeneChip (Affy, NanoString nCounter™ (NanoS, Illumina whole genome RNASeq (RNA-Acc, Illumina targeted RNASeq (t-RNA, and Illumina stranded Total RNA-rRNA-depletion (rRNA. Results Using Affy_FF as the “gold” standard, initial analysis of the 18-gene RAS scores on all 54 samples shows varying pairwise Spearman correlations, with (1 Affy_FFPE (r = 0.233, p = 0.090; (2 NanoS_FFPE (r = 0.608, p < 0.0001; (3 RNA-Acc_FFPE (r = 0.175, p = 0.21; (4 t-RNA_FFPE (r = −0.237, p = 0.085; (5 and t-RNA (r = −0.012, p = 0.93. These results suggest that only NanoString has successful FF to FFPE translation. The subsequent removal of identified “problematic” samples (n = 15 and genes (n = 2 further improves the correlations of Affy_FF with three of the five technologies: Affy_FFPE (r = 0.672, p < 0.0001; NanoS_FFPE (r = 0.738, p < 0.0001; and RNA-Acc_FFPE (r = 0.483, p = 0.002. Conclusions Of the five technology platforms tested, NanoString technology provides a more faithful translation of the RAS pathway gene

Oncogenic K-Ras Activates p38 to Maintain Colorectal Cancer Cell Proliferation during MEK Inhibition

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Winan J. van Houdt

2010-01-01

Full Text Available Background: Colon carcinomas frequently contain activating mutations in the K-ras proto-oncogene. K-ras itself is a poor drug target and drug development efforts have mostly focused on components of the classical Ras-activated MEK/ERK pathway. Here we have studied whether endogenous oncogenic K-ras affects the dependency of colorectal tumor cells on MEK/ERK signaling.

Incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway.

Science.gov (United States)

Takeda, Kosuke; Shao, Danying; Adler, Micha; Charest, Pascale G; Loomis, William F; Levine, Herbert; Groisman, Alex; Rappel, Wouter-Jan; Firtel, Richard A

2012-01-03

Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein-coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase-activating protein acting as a global inhibitor.

The prognostic impact of K-RAS mutations in adult acute myeloid leukemia patients treated with high-dose cytarabine

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Ahmad EI

2011-07-01

Full Text Available Ebtesam I Ahmad, Heba H Gawish, Nashwa MA Al Azizi, Ashraf M ElhefniClinical Pathology Department, Hematology and Oncology Unit of Internal Medicine Department, Faculty of Medicine, Zagazig University, Sharkia, EgyptBackground: Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML. However, little is known about its clinical relevance in the treatment outcome for this leukemia.Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C used in postinduction consolidation chemotherapy in adult AML patients.Patients and methods: The study comprised of 71 de novo AML patients with male/female ratio 1.4:1; their ages ranged from 21–59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G-banding (Giemsa staining, and K-RAS mutation detection using real-time polymerase chain reaction. The patients were randomized into two groups according to the ara-C dose used in consolidation treatment, the high the dose ara-C (HDAC group receiving 400 mg ara-C and-low-dose ara-C (LDAC group receiving 100 mg ara-C; they were followed over a period of five years.Results: Mutations in the K-RAS gene (mutRAS were detected in 23 patients (32% with the remaining 48 patients (68% having wild-type RAS (wtRAS. The percent of blast cells was significantly lower in mutRAS compared to wtRAS patients (P ≤ 0.001 while M4 subtype of AML and Inv(16 frequencies were significantly higher in mutRAS compared to wtRAS patients (P = 0.015 and (P = 0.003, respectively. The patients were followed up for a median of 43 months (range 11–57 months. There was no significant difference in overall survival (OS between mutRAS and wtRAS (P = 0.326. Within the mutRAS

Expression of p53 and p21 in primary glioblastomas

International Nuclear Information System (INIS)

Gross, M.W.; Nashwan, K.; Engenhart-Cabillic, R.; Kraus, A.; Mennel, H.D.; Schlegel, J.

2005-01-01

Background and purpose: primary glioblastomas (GBMs) are highly radioresistant, and in contrast to secondary GBMs, they bear wild-type (wt) p53 protein, which is stabilized in a proportion of these tumors. Therefore, it was investigated in vivo whether p53 expression has prognostic value in patients undergoing radiochemotherapy. Additionally, the authors tried to identify, in vitro, subgroups of primary GBM with different susceptibilities to irradiation, on the basis of their p53 and p21 responses to ionizing radiation. Material and methods: tumor tissue samples from 31 patients suffering from primary GBM undergoing a combined radiochemotherapy with topotecan were investigated. The percentage of cells expressing p53 protein was determined immunohistochemically. Additionally, primary cultures from eleven primary GBMs were established and investigated. p53 and p21 expressions were evaluated before irradiation with 10 Gy and at 2 and 8 h after irradiation. p53 protein expression was measured by western analysis and p21 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). Results: the percentage of p53-positive cells within the tumor specimens obtained from the 31 patients ranged from 0% to 28%, the median value being 4.3%. No significant correlation with disease-free survival or overall survival was found. In vitro, p53 protein was detected in seven of eleven cultures from primary GBM. After irradiation a decrease in p53 protein expression was seen in six of the seven p53-positive cultures. Half of the cultures (two of four) without basal p53 expression showed an increase in p53 expression after irradiation. Basal overexpression of p21 was detected in six of the eleven cultures; in four out of six irradiation led to a decrease in p21 expression. In all cell lines (five of eleven) initially showing absent p21 expression, irradiation induced p21 expression. Despite these responses, G1 arrest was not detectable in any of the GBM cultures

Japāņu mode un subkultūras 20.-21.gadsimtā

OpenAIRE

Murašova, Kristīne

2013-01-01

Bakalaura darba nosaukums ir „Japāņu mode un subkultūras 20.-21.gadsimtā”. Šis darbs sevī iekļauj pētījumu par Japānas populārākajām subkultūrām un modes kustībām pēdējo divu gadsimtu laikā. Pētījums tiek veikts no psiholoģiskā skatu punkta. Darba mērķis ir sniegt lasītājam izpratni par tradicionālas japāņu kultūras konfliktu ar mūsdienu kultūru,analizēt un izprast subkultūru patieso būtību, kā arī uztvert vēstījumu, ko subkultūru pārstāvji cenšas nodot sabiedrībai.Bakalaura darbs sastāv n...

Oncogenic Ras-Induced Morphologic Change Is through MEK/ERK Signaling Pathway to Downregulate Stat3 at a Posttranslational Level in NIH3T3 Cells

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Hsuan-Heng Yeh

2008-01-01

Full Text Available Ras is a key regulator of the MAP kinase-signaling cascade and may cause morphologic change of Ras-transformed cells. Signal transducer and activator of transcription 3 (Stat3 can be activated by cytokine stimulation. In this study, we unravel that Ha-rasV12 overexpression can downregulate the expression of Stat3 protein at a posttranslational level in NIH3T3 cells. Furthermore, we demonstrate that Stat3 expression downregulated by Ha-rasV12 overexpression is through proteosome degradation and not through a mTOR/p70S6K-related signaling pathway. The suppression of Stat3 accompanied by the morphologic change induced by Ha-rasV12 was through mitogen extracellular kinase (MEK/extracellular-regulated kinase (ERK signaling pathway. Microtubule disruption is involved in Ha-rasV12-induced morphologic change, which could be reversed by overexpression of Stat3. Taken together, we are the first to demonstrate that Stat3 protein plays a critical role in Ha-rasV12-induced morphologic change. Oncogenic Ras-triggered morphologic change is through the activation of MEK/ERK to posttranslationally downregulate Stat3 expression. Our finding may shed light on developing novel therapeutic strategies against Ras-related tumorigenesis.

Oxidative Stress Posttranslationally Regulates the Expression of Ha-Ras and Ki-Ras in Cultured Astrocytes

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Samantha Messina

2012-01-01

Full Text Available Addition of hydrogen peroxide to cultured astrocytes induced a rapid and transient increase in the expression of Ha-Ras and Ki-Ras. Pull-down experiments with the GTP-Ras-binding domain of Raf-1 showed that oxidative stress substantially increased the activation of Ha-Ras, whereas a putative farnesylated activated form of Ki-Ras was only slightly increased. The increase in both Ha-Ras and Ki-Ras was insensitive to the protein synthesis inhibitor, cycloheximide, and was occluded by the proteasomal inhibitor, MG-132. In addition, exposure to hydrogen peroxide reduced the levels of ubiquitinated Ras protein, indicating that oxidative stress leads to a reduced degradation of both isoforms through the ubiquitin/proteasome pathway. Indeed, the late reduction in Ha-Ras and Ki-Ras was due to a recovery of proteasomal degradation because it was sensitive to MG-132. The late reduction of Ha-Ras levels was abrogated by compound PD98059, which inhibits the MAP kinase pathway, whereas the late reduction of Ki-Ras was unaffected by PD98059. We conclude that oxidative stress differentially regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes, and that activation of the MAP kinase pathway by oxidative stress itself or by additional factors may act as a fail-safe mechanism limiting a sustained expression of the potentially detrimental Ha-Ras.

The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH

DEFF Research Database (Denmark)

Willumsen, B M; Vass, W C; Velu, T J

1991-01-01

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass...... in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype......., and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming...

ZNF307, a novel zinc finger gene suppresses p53 and p21 pathway

International Nuclear Information System (INIS)

Li Jing; Wang Yuequn; Fan Xiongwei; Mo Xiaoyang; Wang Zequn; Li Yongqing; Yin Zhaochu; Deng Yun; Luo Na; Zhu Chuanbing; Liu Mingyao; Ma Qian; Ocorr, Karen; Yuan Wuzhou; Wu Xiushan

2007-01-01

We have cloned a novel KRAB-related zinc finger gene, ZNF307, encoding a protein of 545 aa. ZNF307 is conserved across species in evolution and is differentially expressed in human adult and fetal tissues. The fusion protein of EGFP-ZNF307 localizes in the nucleus. Transcriptional activity assays show ZNF307 suppresses transcriptional activity of L8G5-luciferase. Overexpressing ZNF307 in different cell lines also inhibits the transcriptional activities of p53 and p21. Moreover, ZNF307 works by reducing the p53 protein level and p53 protein reduction is achieved by increasing transcription of MDM2 and EP300. ZNF307 might suppress p53-p21 pathway through activating MDM2 and EP300 expression and inducing p53 degradation

Inhibitors of Ras-SOS Interactions.

Science.gov (United States)

Lu, Shaoyong; Jang, Hyunbum; Zhang, Jian; Nussinov, Ruth

2016-04-19

Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

p21-ras effector domain mutants constructed by "cassette" mutagenesis

DEFF Research Database (Denmark)

Stone, J C; Vass, W C; Willumsen, B M

1988-01-01

A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

Science.gov (United States)

Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

The effect of S1P receptor signaling pathway on the survival and drug resistance in multiple myeloma cells.

Science.gov (United States)

Fu, Di; Li, Yingchun; Li, Jia; Shi, Xiaoyan; Yang, Ronghui; Zhong, Yuan; Wang, Huihan; Liao, Aijun

2017-01-01

Multiple myeloma (MM) remains incurable by conventional chemotherapy. Sphingosine-1-phosphate (S1P) receptor-mediated signaling has been recently demonstrated to have critical roles in cell survival and drug resistance in a number of hematological malignancies. To dissect the roles of S1P receptor pathway in MM, we systematically examined cell viability and protein expression associated with cell survival and drug resistance in MM cell lines upon treatment with either pathway activator (S1P) or inhibitor (FTY720). Our results reveal that FTY720 inhibits cell proliferation by downregulating expression of target genes, while S1P has an opposite effect. Knocking down of S1P receptor S1P5R results in a reduction of cell survival-related gene expression; however, it does not have impacts on expression of drug resistance genes. These results suggest that S1P signaling plays a role in cell proliferation and drug resistance in MM, and targeting this pathway will provide a new therapeutic direction for MM management.

p55-hGRF, a short natural form of the Ras-GDP exchange factor high yield production and characterization.

Science.gov (United States)

Meyer, P; Janin, J; Baudet-Nessler, S

1999-08-01

p55-hGRF, a natural short form of the guanine-nucleotide-releasing factor for p21-Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39-hGRF. A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates. The recombinant products were resolubilized, renatured and purified to homogeneity. The exchange activity of the refolded hGRF samples on H-Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm. Both p55-hGRF and p39-hGRF form dimers. We established a procedure to prepare and purify the complex with Ras. The results of the characterization study are consistent with a stoichiometry of 1:1 and an equilibrium between dimeric and monomeric forms of the complex.

The Fourth International Symposium on Genetic Disorders of the Ras/MAPK pathway

NARCIS (Netherlands)

D.A. Stevenson (David A.); L. Schill (Lisa); L. Schoyer (Lisa); B.S. Andresen (B.); A. Bakker (Annette); P. Bayrak-Toydemir (Pinar); E.M.M. Burkitt Wright (Emma M.); K. Chatfield (Kathryn); F. Elefteriou (Florent); Y. Elgersma (Ype); M.J. Fisher (Michael J.); D. Franz (David); B.D. Gelb (Bruce); A. Goriely (Anne); K.W. Gripp (Karen); A.Y. Hardan (Antonio Y.); K.M. Keppler-Noreuil (Kim M.); B. Kerr (Bronwyn); B. Korf (Bruce); C. Leoni (Chiara); F. Mccormick (Frank); S.R. Plotkin (Scott R.); K.A. Rauen (Katherine); K. Reilly (Karlyne); A.E. Roberts; A. Sandler (Abby); D. Siegel (Dawn); K.S. Walsh (Karin S.); B.C. Widemann (Brigitte C.)

2016-01-01

textabstractThe RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome,

Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

Science.gov (United States)

Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

2014-01-01

GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization.

Science.gov (United States)

Taylor, Lisa M; James, Alexander; Schuller, Christine E; Brce, Jesena; Lock, Richard B; Mackenzie, Karen L

2004-10-15

Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

TIMP-1 stimulates proliferation of human aortic smooth muscle cells and Ras effector pathways

International Nuclear Information System (INIS)

Akahane, Takemi; Akahane, Manabu; Shah, Amy; Thorgeirsson, Unnur P.

2004-01-01

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein, which is found in most tissues and body fluids. Here, we demonstrated that recombinant TIMP-1 but not the synthetic matrix metalloproteinase inhibitor, GM6001, stimulated proliferation of human aortic smooth muscle cells (AoSMC) in a dose-dependent manner. The mitogenic effect was associated with activation of Ras, increased phosphorylation of ERK, and stimulation of cyclin D1 expression. The phosphatidylinositol 3-kinase (PI3K) signaling pathway was also involved since the PI3K inhibitor, LY294002, abolished the TIMP-1-mediated growth stimulation. These data suggest that TIMP-1 activates Ras, which then turns on the ERK and PI3K signaling pathways to promote cell cycle progression of the AoSMC

CREBBP knockdown enhances RAS/RAF/MEK/ERK signaling in Ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response.

Science.gov (United States)

Dixon, Zach A; Nicholson, Lindsay; Zeppetzauer, Martin; Matheson, Elizabeth; Sinclair, Paul; Harrison, Christine J; Irving, Julie A E

2017-04-01

Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP , are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors. Copyright© Ferrata Storti Foundation.

Molecular analysis of p53 and K-ras in lung carcinomas of coal miners

Energy Technology Data Exchange (ETDEWEB)

Sarkar, F.H.; Li, Y.W.; Vallyathan, V. [Wayne State University, Detroit, MI (United States). School of Medicine, Dept. of Pathology

2001-10-01

Thirty-three cases of non-small cell lung cancers (NSCLC) from the archives of National Coal Workers' Autopsy Study were studied for mutational alterations in p53 and K-ras using PCR-SSCP, DNA sequencing and PCR-oligonucleotide probe hybridization techniques. Mutations of the p53 were observed in 4 smokers (19%) and one in a never smoker (8%). Two polymorphisms in smokers were detected at codon 213, a common site for sequence variation. Among the smokers the p53 mutations were in the heavy smokers. In never smokers there was only a single p53 mutation and two K-ras mutations. In never smokers the frequency of K-ras mutations was similar (17%) in smokers, but one never smoker had two K-ras mutations. Mutations of p53 were more frequent in adenocarcinomas (27%) and they were AT-GC transitions. There were two large cell undifferentiated carcinomas with p53 mutation and one with a K-ras mutation. Two of the 16 squamous cell carcinomas were positive for p53 mutation, while no K-ras mutations were found in this group. The results of these preliminary studies indicate a moderately different mutational spectrum of p53 and K-ras in coal miners independent of cigarette smoking. The mutational spectrum observed in this study of coal miners with heavy cigarette smoking history suggest a protective effect of coal mine dust in preventing abnormal mutations induced by chemical carcinogens in cigarette smoke or reactive oxygen species.

[Effect of ERK1/2 Signaling Pathway Inhibitor PD98059 on the Expression of Ras, BRaf, MEK, ERK1/2 in Marrow Nucleated Red Blood Cells of CMS Patients].

Science.gov (United States)

Han, Yuan-Fang; Ji, Lin-Hua; Feng, Ting-Ting; Liu, Fang; Cui, Sen; Su, Juan

2017-10-01

To investigate the effect of ERK1 / 2 signaling pathway inhibitor PD98059 on Ras, Raf, MEK, ERK1, ERK2 expression in order to explore a new way for basic research and clinical treatment of the chronic mountain sickness(CMS). Sixteen CMS patients were selected, the bone marrow was collected for isolation of monomuclear cells (MNC), the cells were sorted by using CD71 and CD235a antibody magnetic beads, then positive cells were diveded into 5 groups: blank control, DMSO and PD98059 5, 10 and 20 µmol/L, and were cultured in hypoxid condition for 72 hours. The Ras-GTP levels in supernatant was detected by ELISA, the RT-PCR was used to determine the expression of BRaf, MEK, ERK1, ERK2 mRNA in nucleated red blood cells, and the Western blot method was used to detect expression of BRaf, MEK, ERK1, ERK2 protein. PD98059 had no effect on the level of Ras-GTP in each groups. Compared with the blank control group, the expression levels of BRaf, MEK mRNA in DMSO group were not statistically significant (P values were 0.826, 0.298). Compared with the PD98059 20 mol/L group, the expression level of ERK1/2 mRNA was statistically significant (P=0.001, 0.002). Compared with the blank control group, expression levels of p-BRaf, p-MEK protein in DMSO group were not statistically significant (P=0.370, 0.351). Compared with the PD98059 20 mol/L group, the difference of p-ERK1/2 protein level in other 4 groups were statistically significant (P values were <0.001, 0.007). PD98059 can up-regulate the expressions of ERK1/2 miRNA and p-ERK1/2 protein in bone marrow nucleated red blood cells, the Ras / Raf / MEK / ERK 1/2 pathway is the main signal transduction pathway in regulating bone marrow nucleated red blood cells, suggesting that Ras/Raf /MEK /ERK 1/2 pathway may be involved in the pathogenesis of chronic mountain sickness process.

Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

Science.gov (United States)

Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

2017-10-01

Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

Survival pathways under stress

Indian Academy of Sciences (India)

First page Back Continue Last page Graphics. Survival pathways under stress. Bacteria survive by changing gene expression. pattern. Three important pathways will be discussed: Stringent response. Quorum sensing. Proteins performing function to control oxidative damage.

Stat1 phosphorylation determines Ras oncogenicity by regulating p27 kip1.

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Shuo Wang

Full Text Available Inactivation of p27 Kip1 is implicated in tumorigenesis and has both prognostic and treatment-predictive values for many types of human cancer. The transcription factor Stat1 is essential for innate immunity and tumor immunosurveillance through its ability to act downstream of interferons. Herein, we demonstrate that Stat1 functions as a suppressor of Ras transformation independently of an interferon response. Inhibition of Ras transformation and tumorigenesis requires the phosphorylation of Stat1 at tyrosine 701 but is independent of Stat1 phosphorylation at serine 727. Stat1 induces p27 Kip1 expression in Ras transformed cells at the transcriptional level through mechanisms that depend on Stat1 phosphorylation at tyrosine 701 and activation of Stat3. The tumor suppressor properties of Stat1 in Ras transformation are reversed by the inactivation of p27 Kip1. Our work reveals a novel functional link between Stat1 and p27 Kip1, which act in coordination to suppress the oncogenic properties of activated Ras. It also supports the notion that evaluation of Stat1 phosphorylation in human tumors may prove a reliable prognostic factor for patient outcome and a predictor of treatment response to anticancer therapies aimed at activating Stat1 and its downstream effectors.

Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

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Pazman, C; Mayes, C A; Fanto, M; Haynes, S R; Mlodzik, M

2000-04-01

The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

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Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

2018-06-13

The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

[Arnold-Chiari malformation in Noonan syndrome and other syndromes of the RAS/MAPK pathway].

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Ejarque, Ismael; Millán-Salvador, José M; Oltra, Silvestre; Pesudo-Martínez, José V; Beneyto, Magdalena; Pérez-Aytés, Antonio

2015-05-01

Noonan syndrome (NS) and other syndromes with a similar phenotype, such as LEOPARD, cardiofaciocutaneous, Costello and Legius, are associated to mutations in genes included in the RAS/MAPK pathway (RASopathies), which is an important signalling pathway related to cell proliferation. Tonsillar descent into the upper cervical spinal canal, known as Arnold-Chiari malformation (ACM), has been reported in patients with NS and this has led some researchers to suggest that ACM could be part of the phenotypic spectrum of NS. We report two cases of NS and ACM. Case 1: 29-year-old female with Noonan phenotype who underwent surgery at the age of nine years due to pulmonary valve stenosis. At the age of 27, she presented symptomatic ACM that required surgical decompression. She presented the c.922A>G (N308D) mutation in the gene PTPN that belongs to the RAS/MAPK pathway. Case 2: a 10-year-old female with Noonan phenotype and asymptomatic ACM detected in magnetic resonance imaging of the brain. She was a carrier of the c.923A>G (N308S) mutation in gene PTPN11. Six patients with this association have been found in the literature, four with the Noonan phenotype and two with LEOPARD. Our two patients provide supplementary evidence that backs up the hypothesis by which ACM would be part of the phenotypic spectrum of NS. The small number of reported cases of patients with this association does not allow us to draw up recommendations about when and how often neuroimaging studies should be performed; a careful neurological examination, however, should be included in the anticipatory health guidelines in syndromes involving the RAS/MAPK pathway.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

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Yuen, Hiu-Fung; Chan, Ka-Kui; Grills, Claire; Murray, James T.; Platt-Higgins, Angela; Eldin, Osama Sharaf; O’Byrne, Ken; Janne, Pasi; Fennell, Dean A.; Johnston, Patrick G.; Rudland, Philip S.; El-Tanani, Mohamed

2011-01-01

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown compared to normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK and PI3K/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry (PI and Annexin V staining) and MTT assay in cancer cells grown under different conditions after knockdown of Ran.. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. KRas mutated, c-Met amplified and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of KRas or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. PMID:22090358

The freeze-thaw stress response of the yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nutritional state via the RAS-cyclic AMP signal transduction pathway.

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Park, J I; Grant, C M; Attfield, P V; Dawes, I W

1997-10-01

The ability of cells to survive freezing and thawing is expected to depend on the physiological conditions experienced prior to freezing. We examined factors affecting yeast cell survival during freeze-thaw stress, including those associated with growth phase, requirement for mitochondrial functions, and prior stress treatment(s), and the role played by relevant signal transduction pathways. The yeast Saccharomyces cerevisiae was frozen at -20 degrees C for 2 h (cooling rate, less than 4 degrees C min-1) and thawed on ice for 40 min. Supercooling occurred without reducing cell survival and was followed by freezing. Loss of viability was proportional to the freezing duration, indicating that freezing is the main determinant of freeze-thaw damage. Regardless of the carbon source used, the wild-type strain and an isogenic petite mutant ([rho 0]) showed the same pattern of freeze-thaw tolerance throughout growth, i.e., high resistance during lag phase and low resistance during log phase, indicating that the response to freeze-thaw stress is growth phase specific and not controlled by glucose repression. In addition, respiratory ability and functional mitochondria are necessary to confer full resistance to freeze-thaw stress. Both nitrogen and carbon source starvation led to freeze-thaw tolerance. The use of strains affected in the RAS-cyclic AMP (RAS-cAMP) pathway or supplementation of an rca1 mutant (defective in the cAMP phosphodiesterase gene) with cAMP showed that the freeze-thaw response of yeast is under the control of the RAS-cAMP pathway. Yeast did not adapt to freeze-thaw stress following repeated freeze-thaw treatment with or without a recovery period between freeze-thaw cycles, nor could it adapt following pretreatment by cold shock. However, freeze-thaw tolerance of yeast cells was induced during fermentative and respiratory growth by pretreatment with H2O2, cycloheximide, mild heat shock, or NaCl, indicating that cross protection between freeze-thaw stress

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

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Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P ras4B cell growth (P ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

Cost-effectiveness of RAS screening before monoclonal antibodies therapy in metastatic colorectal cancer based on FIRE3 Study

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Wen, Feng; Yang, Yu; Zhang, Pengfei; Zhang, Jian; Zhou, Jing; Tang, Ruilei; Chen, Hongdou; Zheng, Hanrui; Fu, Ping; Li, Qiu

2015-01-01

The surprising results published by FIRE-3 revealed that the overall survival (OS) of RAS wild-type metastatic colorectal cancer (mCRC) patients treated with Cetuximab(Cmab) and FOLFIRI combination was prolonged to 33.1 months. The substantial increase in testing and treatment costs, however, impose a considerable health burden on patients and society. Hence the study was aimed to assess the cost-effectiveness of RAS screening before monoclonal antibodies (mAbs) therapy based on FIRE-3 study. Four groups were analyzed: group 1, patients with KRAS testing treated with Cmab and FOLFIRI; group 2, patients with RAS testing treated with Cmab and FOLFIRI; group 3, patients with KRAS testing treated with bevacizumab(Bmab) and FOLFIRI; group 4, patients with RAS testing treated with Bmab and FOLFIRI. A Markov model comprising 3 health states (progression-free survival, progressive disease and death) was built. The costs were calculated from a Chinese payer perspective, and survival was reported in quality-adjusted life-months (QALMs). Average total lifetime costs ranged from $104,682.44 (RAS-Bmab) to $136,867.44 (RAS-Cmab), while the survival gained varied from 16.88 QALMs in RAS-Bmab to 21.85 QALMs in RAS-Cmab. The cost per QALM was $6,263.86 for RAS-Cmab, $6,145.84 for KRAS-Bmab, $6,201.57 for RAS-Bmab and $6,960.70 for KRAS-Cmab respectively. The KRAS-Cmab strategy was dominated by the other 3 groups. The first-treatment cost of RAS-Cmab was the most influential one to the model. In all, the RAS screening prior to Cmab treatment in mCRC seems to be a cost-effective strategy in the time of monoclonal antibodies (mAbs) therapy with the most gained QALMs. PMID:26418570

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

Science.gov (United States)

Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2014-03-04

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

Science.gov (United States)

Deng, Wen; Zhou, Yuan; Tiwari, Agnes FY; Su, Hang; Yang, Jie; Zhu, Dandan; Lau, Victoria Ming Yi; Hau, Pok Man; Yip, Yim Ling; Cheung, Annie LM; Guan, Xin-Yuan; Tsao, Sai Wah

2015-01-01

Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21. PMID:25131797

Ras signaling in aging and metabolic regulation.

Science.gov (United States)

Slack, Cathy

2017-12-07

Aberrant signal transduction downstream of the Ras GTPase has a well-established role in tumorigenesis. Mutations that result in hyperactivation of Ras are responsible for a third of all human cancers. Hence, small molecule inhibitors of the Ras signal transduction cascade have been under intense focus as potential cancer treatments. In both invertebrate and mammalian models, emerging evidence has also implicated components of the Ras signaling pathway in aging and metabolic regulation. Here, I review the current evidence for Ras signaling in these newly discovered roles highlighting the interactions between the Ras pathway and other longevity assurance mechanisms. Defining the role of Ras signaling in maintaining age-related health may have important implications for the development of interventions that could not only increase lifespan but also delay the onset and/or progression of age-related functional decline.

K-RAS and N-RAS mutations in testicular germ cell tumors

Directory of Open Access Journals (Sweden)

Bekir Muhammet Hacioglu

2017-05-01

Full Text Available Testicular cancer is a relatively rare tumor type, accounting for approximately 1% of all cancers in men. However, among men aged between 15 and 40 years, testicular cancer is the most commonly diagnosed malignancy. Testicular germ cell tumors (TGCTs are classified as seminoma and non-seminoma. The RAS oncogene controls several cellular functions, including cell proliferation, apoptosis, migration, and differentiation. Thus, RAS signaling is important for normal germ cell development. Mutations of the Kirsten RAS (K-RAS gene are present in over 20% of all cancers. RAS gene mutations have also been reported in TGCTs. We investigated K-RAS and N-RAS mutations in seminoma and non-seminoma TGCT patients. A total of 24 (55% pure seminoma cases and 19 (45% non-seminoma cases were included in the study. K-RAS and N-RAS analyses were performed in our molecular pathology laboratory, using K-RAS and N-RAS Pyro Kit 24 V1 (Qiagen. In total, a RAS mutation was present in 12 patients (27%: 7 seminoma (29% and 5 non-seminoma cases (26% [p = 0.55]. A K-RAS mutation was present in 4 pure seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63], and an N-RAS mutation was observed in 4 seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63]. Both, K-RAS and N-RAS mutations were present in two patients: one with seminoma tumor and the other with non-seminoma tumor. To date, no approved targeted therapy is available for the treatment of TGCTs. The analysis of K-RAS and N-RAS mutations in these tumors may provide more treatment options, especially in platinum-resistant tumors.

Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway.

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Bissinger, P H; Wieser, R; Hamilton, B; Ruis, H

1989-03-01

In Saccharomyces cerevisiae, lack of nutrients triggers a pleiotropic response characterized by accumulation of storage carbohydrates, early G1 arrest, and sporulation of a/alpha diploids. This response is thought to be mediated by RAS proteins, adenylate cyclase, and cyclic AMP (cAMP)-dependent protein kinases. This study shows that expression of the S. cerevisiae gene coding for a cytoplasmic catalase T (CTT1) is controlled by this pathway: it is regulated by the availability of nutrients. Lack of a nitrogen, sulfur, or phosphorus source causes a high-level expression of the gene. Studies with strains with mutations in the RAS-cAMP pathway and supplementation of a rca1 mutant with cAMP show that CTT1 expression is under negative control by a cAMP-dependent protein kinase and that nutrient control of CTT1 gene expression is mediated by this pathway. Strains containing a CTT1-Escherichia coli lacZ fusion gene have been used to isolate mutants with mutations in the pathway. Mutants characterized in this investigation fall into five complementation groups. Both cdc25 and ras2 alleles were identified among these mutants.

CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways

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Schulte, Simone Laura; Waha, Andreas; Steiger, Barbara; Denkhaus, Dorota; Dörner, Evelyn; Calaminus, Gabriele; Leuschner, Ivo; Pietsch, Torsten

2016-01-01

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy. PMID:27391150

ERK mediated upregulation of death receptor 5 overcomes the lack of p53 functionality in the diaminothiazole DAT1 induced apoptosis in colon cancer models: efficiency of DAT1 in Ras-Raf mutated cells.

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Thamkachy, Reshma; Kumar, Rohith; Rajasekharan, K N; Sengupta, Suparna

2016-03-08

p53 is a tumour suppressor protein that plays a key role in many steps of apoptosis, and malfunctioning of this transcription factor leads to tumorigenesis. Prognosis of many tumours also depends upon the p53 status. Most of the clinically used anticancer compounds activate p53 dependent pathway of apoptosis and hence require p53 for their mechanism of action. Further, Ras/Raf/MEK/ERK axis is an important signaling pathway activated in many cancers. Dependence of diaminothiazoles, compounds that have gained importance recently due to their anticancer and anti angiogenic activities, were tested in cancer models with varying p53 or Ras/Raf mutational status. In this study we have used p53 mutated and knock out colon cancer cells and xenograft tumours to study the role of p53 in apoptosis mediated by diaminothiazoles. Colon cancer cell lines with varying mutational status for Ras or Raf were also used. We have also examined the toxicity and in vivo efficacy of a lead diaminothiazole 4-Amino-5-benzoyl-2-(4-methoxy phenylamino)thiazole (DAT1) in colon cancer xenografts. We have found that DAT1 is active in both in vitro and in vivo models with nonfunctional p53. Earlier studies have shown that extrinsic pathway plays major role in DAT1 mediated apoptosis. In this study, we have found that DAT1 is causing p53 independent upregulation of the death receptor 5 by activating the Ras/Raf/MEK/ERK signaling pathway both in wild type and p53 suppressed colon cancer cells. These findings are also confirmed by the in vivo results. Further, DAT1 is more efficient to induce apoptosis in colon cancer cells with mutated Ras or Raf. Minimal toxicity in both acute and subacute studies along with the in vitro and in vivo efficacy of DAT1 in cancers with both wild type and nonfunctional p53 place it as a highly beneficial candidate for cancer chemotherapy. Besides, efficiency in cancer cells with mutations in the Ras oncoprotein or its downstream kinase Raf raise interest in

Cell survival, cell death and cell cycle pathways are interconnected: Implications for cancer therapy

DEFF Research Database (Denmark)

Maddika, S; Ande, SR; Panigrahi, S

2007-01-01

)), and the Cip1/Waf1/Kip1-2-family (p21(Cip1/Waf1), p27(Kip1), p57(Kip2)) are shown both in the context of proliferation regulators and as contributors to the apoptotic machinery. Bcl2-family members (i.e. Bcl2, Bcl-X(L) Mcl-1(L); Bax, Bok/Mtd, Bak, and Bcl-X(S); Bad, Bid, Bim(EL), Bmf, Mcl-1(S)) are highlighted...... approaches that would involve redirecting over-active survival and proliferation pathways towards induction of apoptosis in cancer cells....

Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

OpenAIRE

Baltus, E; Hanocq-Quertier, J; Hanocq, F; Brachet, J

1988-01-01

The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the ...

Genetic alterations in Ki-ras and Ha-ras genes in Juvenile Nasopharyngeal Angiofibromas and head and neck cancer

Directory of Open Access Journals (Sweden)

Cláudia Malheiros Coutinho

1999-05-01

Full Text Available CONETXT: Ras gene mutations have been associated to a wide range of human solid tumors. Members of the ras gene family (Ki-ras, Ha-ras and N-ras are structurally related and code for a protein (p21 known to play an important role in the regulation of normal signal transduction and cell growth. The frequency of ras mutations is different from one type of tumor to another, suggesting that point mutations might be carcinogen-specific. OBJECTIVES: To study the occurrence of Ki-ras and Ha-ras mutations. We also studied the relative level of Ha-ras mRNA in 32 of the head and neck tumors. DESIGN: Case series. SETTING: University referral unit. PARTICIPANTS: 60 head and neck tumors and in 28 Juvenile Nasopharyngeal Angiofibromas (JNA. DIAGNOSTIC TEST: Using PCR-SSCP we examined the occurrence of Ki-ras and Ha-ras mutations. The relative level of Ha-ras mRNA was examined by Northern blot analysis. RESULTS: None of the head and neck tumors or JNA samples showed evidence of mutations within codons 12, 13, 59 and 61 of Ki-ras or Ha-ras genes. However, 17 (53% of the tumors where gene expression could be examined exhibited increased levels of Ha-ras mRNA compared with the normal tissue derived from the same patient. CONCLUSIONS: Our results demonstrate for the first time that mutations of Ki-ras and Ha-ras genes are not associated with the development of JNA and confirm previous reports indicating that activating ras mutations are absent or rarely involved in head and neck tumors from western world patients. Furthermore, our findings suggest that overexpression of Ha-ras, rather than mutations, might be an important factor in the development and progression of head and neck tumors.

The CDK inhibitor p21 is a novel target gene of ATF4 and contributes to cell survival under ER stress.

Science.gov (United States)

Inoue, Yasumichi; Kawachi, Shiori; Ohkubo, Tsubasa; Nagasaka, Mai; Ito, Shogo; Fukuura, Keishi; Itoh, Yuka; Ohoka, Nobumichi; Morishita, Daisuke; Hayashi, Hidetoshi

2017-11-01

Activating transcription factor 4 (ATF4) is well known for its role in the endoplasmic reticulum (ER) stress response. ATF4 also transcriptionally induces multiple effectors that determine cell fate depending on cellular context. In addition, ATF4 can communicate both pro-apoptotic and pro-survival signals. How ATF4 mediates its prosurvival roles, however, requires further investigation. Here, we report that the CDK inhibitor p21 is a novel target gene of ATF4. We identified two ATF4-responsive elements, one of which directly binds ATF4, within the first intron of the p21 gene. Importantly, overexpression of p21 enhances cell survival following ER stress induction, while p21 knockdown increases cell death. These results suggest that p21 induction plays a vital role in the cellular response to ER stress and indicate that p21 is a prosurvival effector of ATF4. © 2017 Federation of European Biochemical Societies.

Resistance and resilience of small-scale recirculating aquaculture systems (RAS) with or without algae to pH perturbation

Science.gov (United States)

Giatsis, Christos; Md Yusoff, Fatimah; Verreth, Johan; Verdegem, Marc

2018-01-01

The experimental set-up of this study mimicked recirculating aquaculture systems (RAS) where water quality parameters such as dissolved oxygen, pH, temperature, and turbidity were controlled and wastes produced by fish and feeding were converted to inorganic forms. A key process in the RAS was the conversion of ammonia to nitrite and nitrite to nitrate through nitrification. It was hypothesized that algae inclusion in RAS would improve the ammonia removal from the water; thereby improving RAS water quality and stability. To test this hypothesis, the stability of the microbiota community composition in a freshwater RAS with (RAS+A) or without algae (RAS-A) was challenged by introducing an acute pH drop (from pH 7 to 4 during three hours) to the system. Stigeoclonium nanum, a periphytic freshwater microalga was used in this study. No significant effect of the algae presence was found on the resistance to the acute pH drop on ammonia conversion to nitrite and nitrite conversion to nitrate. Also the resilience of the ammonia conversion to the pH drop disruption was not affected by the addition of algae. This could be due to the low biomass of algae achieved in the RAS. However, with regard to the conversion step of nitrite to nitrate, RAS+A was significantly more resilient than RAS-A. In terms of overall bacterial communities, the composition and predictive function of the bacterial communities was significantly different between RAS+A and RAS-A. PMID:29659617

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling.

Science.gov (United States)

Erickson, Keesha E; Rukhlenko, Oleksii S; Posner, Richard G; Hlavacek, William S; Kholodenko, Boris N

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisition of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers. Copyright © 2018 Elsevier Ltd. All rights reserved.

The third international meeting on genetic disorders in the RAS/MAPK pathway: towards a therapeutic approach.

Science.gov (United States)

Korf, Bruce; Ahmadian, Reza; Allanson, Judith; Aoki, Yoko; Bakker, Annette; Wright, Emma Burkitt; Denger, Brian; Elgersma, Ype; Gelb, Bruce D; Gripp, Karen W; Kerr, Bronwyn; Kontaridis, Maria; Lazaro, Conxi; Linardic, Corinne; Lozano, Reymundo; MacRae, Calum A; Messiaen, Ludwine; Mulero-Navarro, Sonia; Neel, Benjamin; Plotkin, Scott; Rauen, Katherine A; Roberts, Amy; Silva, Alcino J; Sittampalam, Sitta G; Zhang, Chao; Schoyer, Lisa

2015-08-01

"The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach" was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion. © 2015 Wiley Periodicals, Inc.

Prognostic implications of molecular and immunohistochemical profiles of the Rb and p53 cell cycle regulatory pathways in primary non-small cell lung carcinoma.

LENUS (Irish Health Repository)

Burke, Louise

2012-02-03

PURPOSE: Many studies have highlighted the aberrant expression and prognostic significance of individual proteins in either the Rb (particularly cyclin D1, p16INK4A, and pRb) or the p53 (p53 and p21Waf1) pathways in non-small cell lung cancer. We hypothesize that cumulative abnormalities within each and between these pathways would have significant prognostic potential regarding survival. EXPERIMENTAL DESIGN: Our study population consisted of 106 consecutive surgically resected cases of predominantly early-stage non-small cell lung cancer from the National Cancer Institute-Mayo Clinic series, and assessment of proteins involved both immunohistochemical (cyclin D1, p21Waf1, pRb, p16INK4A, and p53) and mutational analysis (p53) in relationship to staging and survival. RESULTS: Cyclin D1 overexpression was noted in 48% of the tumors, p16INK4A negative in 53%, pRb negative in 17%, p53 immunopositive in 50%, p53 mutation frequency in 48%, and p21(Waf1) overexpression in 47%, none with prognostic significance. Cyclin D1 overexpression in pRb-negative tumors revealed a significantly worse prognosis with a mean survival of 2.3 years (P = 0.004). A simultaneous p53 mutation dramatically reduced the mean survival time to 0.9 years (P = 0.007). Cyclin D1 overexpression with either a p53 mutation or a p53 overexpression was also associated with a significantly poorer prognosis (P = 0.0033 and 0.0063, respectively). CONCLUSIONS: Some cumulative abnormalities in the Rb and p53 pathways (e.g., cyclin D1 overexpression and p53 mutations) significantly cooperate to predict a poor prognosis; however, the complexity of the cell cycle protein interaction in any given tumor warrants caution in interpreting survival results when specific protein abnormalities are taken in isolation.

Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

Energy Technology Data Exchange (ETDEWEB)

Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

2012-04-15

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.

Science.gov (United States)

Blum, Roy; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Kloog, Yoel

2006-09-01

The Ras inhibitor farnesylthiosalicylic acid (FTS) has been shown to induce apoptosis in glioblastoma multiforme, but its mechanism of action was unknown. We show that FTS or dominant-negative Ras, by deregulating extracellular signal-regulated kinase and Akt signaling, decreases survivin gene transcripts in U87 glioblastoma multiforme, leading to disappearance of survivin protein and cell death. FTS affected both Ras-controlled regulators of survivin transcription and Ras-regulated survival signals. Thus, Ras inhibition by FTS resulted in release of the survivin "brake" on apoptosis and in activation of the mitochondrial apoptotic pathway: dephosphorylation of Bad, activation of Bax, release of cytochrome c, and caspase activation. FTS-induced apoptosis of U87 cells was strongly attenuated by forced expression of survivin or by caspase inhibitors. These results show that resistance to apoptosis in glioblastoma multiforme can be abolished by a single Ras inhibitor, which targets both survivin, a critical inhibitor of apoptosis, and the intrinsic mitochondrial apoptotic machinery.

Downregulation of B-myb promotes senescence via the ROS-mediated p53/p21 pathway, in vascular endothelial cells.

Science.gov (United States)

Zhou, Zhihui; Yin, Yanlin; Chang, Qun; Sun, Guanqun; Lin, Jiahui; Dai, Yalei

2017-04-01

To reveal whether B-myb is involved in preventing senescence of vascular endothelial cells, and if so, to identify possible mechanisms for it. C57/BL6 male mice and primary human aortic endothelial cells (HAECs) were used. Bleomycin was applied to induce stress-related premature senescence. B-myb knockdown was achieved using an siRNA technique and cell senescence was assessed using the senescence-associated β-galactosidase (SA-β-gal) assay. Intracellular reactive oxygen species (ROS) production was analysed using an ROS assay kit and cell proliferation was evaluated using KFluor488 EdU kit. Capillary tube network formation was determined by Matrigel assay. Expressions of mRNA and protein levels were detected by real-time PCR and western blotting. B-myb expression significantly decreased, while p53 and p21 expressions increased in the aortas of aged mice. This expression pattern was also found in replicative senescent HAECs and senescent HAECs induced by bleomycin. B-myb knockdown resulted in upregulation of p22 phox , ROS accumulation and cell senescence of HAECs. Downregulation of B-myb significantly inhibited cell proliferation and capillary tube network formation and activated the p53/p21 signalling pathway. Blocking ROS production or inhibiting p53 activation remarkably attenuated SA-β-gal activity and delayed cell senescence induced by B-myb-silencing. Downregulation of B-myb induced senescence by upregulation of p22 phox and activation of the ROS/p53/p21 pathway, in our vascular endothelial cells, suggesting that B-myb may be a novel candidate for regulating cell senescence to protect against endothelial senescence-related cardiovascular diseases. © 2016 John Wiley & Sons Ltd.

Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms

International Nuclear Information System (INIS)

Mackenzie, Robertson; Kommoss, Stefan; Winterhoff, Boris J.; Kipp, Benjamin R.; Garcia, Joaquin J.; Voss, Jesse; Halling, Kevin; Karnezis, Anthony; Senz, Janine; Yang, Winnie; Prigge, Elena-Sophie; Reuschenbach, Miriam; Doeberitz, Magnus Von Knebel; Gilks, Blake C.; Huntsman, David G.; Bakkum-Gamez, Jamie; McAlpine, Jessica N.; Anglesio, Michael S.

2015-01-01

Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be “RAS-pathway alteration negative”, using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell

Alterations in the K-ras and p53 genes in rat lung tumors

Energy Technology Data Exchange (ETDEWEB)

Belinsky, S.A.; Swafford, D.S.; Finch, G.L.; Mitchell, C.E. [Inhalation Toxicology Research Institute, Albuquerque, NM (United States)] [and others

1997-06-01

Activation of the K-ras protooncogene and inactivation of the p53 tumor suppressor gene are events common to many types of human cancers. Molecular epidemiology studies have associated mutational profiles in these genes with specific exposures. The purpose of this paper is to review investigations that have examined the role of the K-ras and p53 genes in lung tumors induced in the F344 rat by mutagenic and nonmutagenic exposures. Mutation profiles within the K-ras and p53 genes, if present in rat lung tumors, would help to define some of the molecular mechanisms underlying cancer induction by various environmental agents. Pulmonary adenocarcinomas or squamous cell carcinomas were induced by tetranitromethane (TNM), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), beryllium metal, plutonium-239, X-ray, diesel exhaust, or carbon black. These agents were chosen because the tumors they produced could arise via different types of DNA damage. Mutation of the K-ras gene was determined by approaches that included DNA transfection, direct sequencing, mismatch hybridization, and restriction fragment length polymorphism analysis. The frequency for mutation of the K-ras gene was exposure dependent. The transition mutations formed could have been derived from deamination of cytosine. Alteration in the p53 gene was assessed by immunohistochemical analysis for p53 protein and single-strand conformation polymorphism (SSCP) analysis of exons 4 to 9. None of the 93 adenocarinomas examined was immunoreactive toward the anti-p53 antibody CM1. In contrast, 14 of 71 squamous cell carcinomas exhibited nuclear p53 immunoreactivity with no correlation to type of exposure. However, SSCP analysis only detected mutations in 2 of 14 squamous cell tumors that were immunoreactive, suggesting that protein stabilization did not stem from mutations within the p53 gene. Thus, the p53 gene does not appear to be involved in the genesis of most rat lung tumors. 2 figs., 2 tabs., 48 refs.

Survival associated pathway identification with group Lp penalized global AUC maximization

Directory of Open Access Journals (Sweden)

Liu Zhenqiu

2010-08-01

Full Text Available Abstract It has been demonstrated that genes in a cell do not act independently. They interact with one another to complete certain biological processes or to implement certain molecular functions. How to incorporate biological pathways or functional groups into the model and identify survival associated gene pathways is still a challenging problem. In this paper, we propose a novel iterative gradient based method for survival analysis with group Lp penalized global AUC summary maximization. Unlike LASSO, Lp (p 1. We first extend Lp for individual gene identification to group Lp penalty for pathway selection, and then develop a novel iterative gradient algorithm for penalized global AUC summary maximization (IGGAUCS. This method incorporates the genetic pathways into global AUC summary maximization and identifies survival associated pathways instead of individual genes. The tuning parameters are determined using 10-fold cross validation with training data only. The prediction performance is evaluated using test data. We apply the proposed method to survival outcome analysis with gene expression profile and identify multiple pathways simultaneously. Experimental results with simulation and gene expression data demonstrate that the proposed procedures can be used for identifying important biological pathways that are related to survival phenotype and for building a parsimonious model for predicting the survival times.

The inflammatory cytokine TNFα cooperates with Ras in elevating metastasis and turns WT-Ras to a tumor-promoting entity in MCF-7 cells

International Nuclear Information System (INIS)

Leibovich-Rivkin, Tal; Liubomirski, Yulia; Meshel, Tsipi; Abashidze, Anastasia; Brisker, Daphna; Solomon, Hilla; Rotter, Varda; Weil, Miguel; Ben-Baruch, Adit

2014-01-01

In the present study we determined the relative contribution of two processes to breast cancer progression: (1) Intrinsic events, such as activation of the Ras pathway and down-regulation of p53; (2) The inflammatory cytokines TNFα and IL-1β, shown in our published studies to be highly expressed in tumors of >80% of breast cancer patients with recurrent disease. Using MCF-7 human breast tumor cells originally expressing WT-Ras and WT-p53, we determined the impact of the above-mentioned elements and cooperativity between them on the expression of CXCL8 (ELISA, qRT-PCR), a member of a “cancer-related chemokine cluster” that we have previously identified. Then, we determined the mechanisms involved (Ras-binding-domain assays, Western blot, luciferase), and tested the impact of Ras + TNFα on angiogenicity (chorioallantoic membrane assays) and on tumor growth at the mammary fat pad of mice and on metastasis, in vivo. Using Ras G12V that recapitulates multiple stimulations induced by receptor tyrosine kinases, we found that Ras G12V alone induced CXCL8 expression at the mRNA and protein levels, whereas down-regulation of p53 did not. TNFα and IL-1β potently induced CXCL8 expression and synergized with Ras G12V , together leading to amplified CXCL8 expression. Testing the impact of WT-Ras, which is the common form in breast cancer patients, we found that WT-Ras was not active in promoting CXCL8; however, TNFα has induced the activation of WT-Ras: joining these two elements has led to cooperative induction of CXCL8 expression, via the activation of MEK, NF-κB and AP-1. Importantly, TNFα has led to increased expression of WT-Ras in an active GTP-bound form, with properties similar to those of Ras G12V . Jointly, TNFα + Ras activities have given rise to increased angiogenesis and to elevated tumor cell dissemination to lymph nodes. TNFα cooperates with Ras in promoting the metastatic phenotype of MCF-7 breast tumor cells, and turns WT-Ras into a tumor

Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

Directory of Open Access Journals (Sweden)

Fiorella Guadagni

2012-01-01

Full Text Available The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala of exon 1. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein

International Nuclear Information System (INIS)

Lee, G.; Ronai, Z.A.; Pincus, M.R.; Brandt-Rauf, P.W.; Weinstein, I.B.; Murphy, R.B.; Delohery, T.M.; Nishimura, S.; Yamaizumi, Z.

1989-01-01

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with modified p21 protein, the cells were pulsed with [ 35 S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21 - protein complexes. By using this technique, the authors found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. They suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes

The Prognostic Impact of p53 Expression on Sporadic Colorectal Cancer Is Dependent on p21 Status

International Nuclear Information System (INIS)

Kruschewski, Martin; Mueller, Kathrin; Lipka, Sybille; Budczies, Jan; Noske, Aurelia; Buhr, Heinz Johannes; Elezkurtaj, Sefer

2011-01-01

The prognostic value of p53 and p21 expression in colorectal cancer is still under debate. We hypothesize that the prognostic impact of p53 expression is dependent on p21 status. The expression of p53 and p21 was immunohistochemically investigated in a prospective cohort of 116 patients with UICC stage II and III sporadic colorectal cancer. The results were correlated with overall and recurrence-free survival. The mean observation period was 51.8 ± 2.5 months. Expression of p53 was observed in 72 tumors (63%). Overall survival was significantly better in patients with p53-positive carcinomas than in those without p53 expression (p = 0.048). No differences were found in recurrence-free survival (p = 0.161). The p53+/p21− combination was seen in 68% (n = 49), the p53+/p21+ combination in 32% (n = 23). Patients with p53+/p21− carcinomas had significantly better overall and recurrence-free survival than those with p53+/p21+ (p < 0.0001 resp. p = 0.003). Our data suggest that the prognostic impact of p53 expression on sporadic colorectal cancer is dependent on p21 status

DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.

Science.gov (United States)

Watanabe-Takano, Haruko; Takano, Kazunori; Hatano, Masahiko; Tokuhisa, Takeshi; Endo, Takeshi

2015-01-01

Myofibroblasts play critical roles in the development of idiopathic pulmonary fibrosis by depositing components of extracellular matrix. One source of lung myofibroblasts is thought to be alveolar epithelial type 2 cells that undergo epithelial-mesenchymal transition (EMT). Rat RLE-6TN alveolar epithelial type 2 cells treated with transforming growth factor-β1 (TGF-β1) are converted into myofibroblasts through EMT. TGF-β induces both canonical Smad signaling and non-canonical signaling, including the Ras-induced ERK pathway (Raf-MEK-ERK). However, the signaling mechanisms regulating TGF-β1-induced EMT are not fully understood. Here, we show that the Ras-ERK pathway negatively regulates TGF-β1-induced EMT in RLE-6TN cells and that DA-Raf1 (DA-Raf), a splicing isoform of A-Raf and a dominant-negative antagonist of the Ras-ERK pathway, plays an essential role in EMT. Stimulation of the cells with fibroblast growth factor 2 (FGF2), which activated the ERK pathway, prominently suppressed TGF-β1-induced EMT. An inhibitor of MEK, but not an inhibitor of phosphatidylinositol 3-kinase, rescued the TGF-β1-treated cells from the suppression of EMT by FGF2. Overexpression of a constitutively active mutant of a component of the Ras-ERK pathway, i.e., H-Ras, B-Raf, or MEK1, interfered with EMT. Knockdown of DA-Raf expression with siRNAs facilitated the activity of MEK and ERK, which were only weakly and transiently activated by TGF-β1. Although DA-Raf knockdown abrogated TGF-β1-induced EMT, the abrogation of EMT was reversed by the addition of the MEK inhibitor. Furthermore, DA-Raf knockdown impaired the TGF-β1-induced nuclear translocation of Smad2, which mediates the transcription required for EMT. These results imply that intrinsic DA-Raf exerts essential functions for EMT by antagonizing the TGF-β1-induced Ras-ERK pathway in RLE-6TN cells.

Compound K, a Ginsenoside Metabolite, Inhibits Colon Cancer Growth via Multiple Pathways Including p53-p21 Interactions

Directory of Open Access Journals (Sweden)

Eugene B. Chang

2013-01-01

Full Text Available Compound K (20-O-beta-D-glucopyranosyl-20(S-protopanaxadiol, CK, an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of cancers. However, the mechanisms are not completely understood, especially in colorectal cancer (CRC. A xenograft tumor model was used first to examine the anti-CRC effect of CK in vivo. Then, multiple in vitro assays were applied to investigate the anticancer effects of CK including antiproliferation, apoptosis and cell cycle distribution. In addition, a qPCR array and western blot analysis were executed to screen and validate the molecules and pathways involved. We observed that CK significantly inhibited the growth of HCT-116 tumors in an athymic nude mouse xenograft model. CK significantly inhibited the proliferation of human CRC cell lines HCT-116, SW-480, and HT-29 in a dose- and time-dependent manner. We also observed that CK induced cell apoptosis and arrested the cell cycle in the G1 phase in HCT-116 cells. The processes were related to the upregulation of p53/p21, FoxO3a-p27/p15 and Smad3, and downregulation of cdc25A, CDK4/6 and cyclin D1/3. The major regulated targets of CK were cyclin dependent inhibitors, including p21, p27, and p15. These results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in CRC, suggesting that CK could be an active compound in the prevention or treatment of CRC.

Metformin and Resveratrol Inhibited High Glucose-Induced Metabolic Memory of Endothelial Senescence through SIRT1/p300/p53/p21 Pathway.

Science.gov (United States)

Zhang, Erli; Guo, Qianyun; Gao, Haiyang; Xu, Ruixia; Teng, Siyong; Wu, Yongjian

2015-01-01

Endothelial senescence plays crucial roles in diabetic vascular complication. Recent evidence indicated that transient hyperglycaemia could potentiate persistent diabetic vascular complications, a phenomenon known as "metabolic memory." Although SIRT1 has been demonstrated to mediate high glucose-induced endothelial senescence, whether and how "metabolic memory" would affect endothelial senescence through SIRT1 signaling remains largely unknown. In this study, we investigated the involvement of SIRT1 axis as well as the protective effects of resveratrol (RSV) and metformin (MET), two potent SIRT1 activators, during the occurrence of "metabolic memory" of cellular senescence (senescent "memory"). Human umbilical vascular endothelial cells (HUVECs) were cultured in either normal glucose (NG)/high glucose (HG) media for 6 days, or 3 days of HG followed by 3 days of NG (HN), with or without RSV or MET treatment. It was shown that HN incubation triggered persistent downregulation of deacetylase SIRT1 and upregulation of acetyltransferase p300, leading to sustained hyperacetylation (at K382) and activation of p53, and subsequent p53/p21-mediated senescent "memory." In contrast, senescent "memory" was abrogated by overexpression of SIRT1 or knockdown of p300. Interestingly, we found that SIRT1 and p300 could regulate each other in response to HN stimulation, suggesting that a delicate balance between acetyltransferases and deacetylases may be particularly important for sustained acetylation and activation of non-histone proteins (such as p53), and eventually the occurrence of "metabolic memory." Furthermore, we found that RSV or MET treatment prevented senescent "memory" by modulating SIRT1/p300/p53/p21 pathway. Notably, early and continuous treatment of MET, but not RSV, was particularly important for preventing senescent "memory." In conclusion, short-term high glucose stimulation could induce sustained endothelial senescence via SIRT1/p300/p53/p21 pathway. RVS or MET

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein.

Science.gov (United States)

Lee, G; Ronai, Z A; Pincus, M R; Brandt-Rauf, P W; Murphy, R B; Delohery, T M; Nishimura, S; Yamaizumi, Z; Weinstein, I B

1989-11-01

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

Cyclophilin B Supports Myc and Mutant p53 Dependent Survival of Glioblastoma Multiforme Cells

Science.gov (United States)

Choi, Jae Won; Schroeder, Mark A.; Sarkaria, Jann N.; Bram, Richard J.

2014-01-01

Glioblastoma multiforme (GBM) is an aggressive, treatment-refractory type of brain tumor for which effective therapeutic targets remain important to identify. Here we report that cyclophilin B (CypB), a prolyl isomerase residing in the endoplasmic reticulum (ER), provides an essential survival signal in GBM cells. Analysis of gene expression databases revealed that CypB is upregulated in many cases of malignant glioma. We found that suppression of CypB reduced cell proliferation and survival in human GBM cells in vitro and in vivo. We also found that treatment with small molecule inhibitors of cyclophilins, including the approved drug cyclosporine, greatly reduced the viability of GBM cells. Mechanistically, depletion or pharmacologic inhibition of CypB caused hyperactivation of the oncogenic RAS-MAPK pathway, induction of cellular senescence signals, and death resulting from loss of MYC, mutant p53, Chk1 and JAK/STAT3 signaling. Elevated reactive oxygen species, ER expansion and abnormal unfolded protein responses in CypB-depleted GBM cells indicated that CypB alleviates oxidative and ER stresses and coordinates stress adaptation responses. Enhanced cell survival and sustained expression of multiple oncogenic proteins downstream of CypB may thus contribute to the poor outcome of GBM tumors. Our findings link chaperone-mediated protein folding in the ER to mechanisms underlying oncogenic transformation, and they make CypB an attractive and immediately targetable molecule for GBM therapy. PMID:24272483

Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

Science.gov (United States)

Severyn, Bryan; Nguyen, Thi; Altman, Michael D; Li, Lixia; Nagashima, Kumiko; Naumov, George N; Sathyanarayanan, Sriram; Cook, Erica; Morris, Erick; Ferrer, Marc; Arthur, Bill; Benita, Yair; Watters, Jim; Loboda, Andrey; Hermes, Jeff; Gilliland, D Gary; Cleary, Michelle A; Carroll, Pamela M; Strack, Peter; Tudor, Matt; Andersen, Jannik N

2016-10-01

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway. © 2016 Society for Laboratory Automation and Screening.

Activation of RAS/ERK alone is insufficient to inhibit RXRα function and deplete retinoic acid in hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Wang, Ai-Guo, E-mail: wangaiguotl@hotmail.com; Song, Ya-Nan; Chen, Jun; Li, Hui-Ling; Dong, Jian-Yi; Cui, Hai-Peng; Yao, Liang; Li, Xue-Feng; Gao, Wen-Ting; Qiu, Ze-Wen; Wang, Fu-Jin; Wang, Jing-Yu, E-mail: wangjingyus@163.com

2014-09-26

Highlights: • The activation of RAS/ERK is insufficient to inhibit RXRα function and deplete RA. • The retinoid metabolism-related genes are down-regulated by ras oncogene. • The atRA has no effect on preventing hepatic tumorigenesis or curing the developed hepatic nodules. - Abstract: Activation of RAS/ERK signaling pathway, depletion of retinoid, and phosphorylation of retinoid X receptor alpha (RXRα) are frequent events found in liver tumors and thought to play important roles in hepatic tumorigenesis. However, the relationships among them still remained to be elucidated. By exploring the transgenic mouse model of hepatic tumorigenesis induced by liver-specific expression of H-ras12V oncogene, the activation of RAS/ERK, the mRNA expression levels of retinoid metabolism-related genes, the contents of retinoid metabolites, and phosphorylation of RXRα were determined. RAS/ERK signaling pathway was gradually and significantly activated in hepatic tumor adjacent normal liver tissues (P) and hepatic tumor tissues (T) of H-ras12V transgenic mice compared with normal liver tissues (Wt) of wild type mice. On the contrary, the mRNA expression levels of retinoid metabolism-related genes were significantly reduced in P and T compared with Wt. Interestingly, the retinoid metabolites 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (atRA), the well known ligands for nuclear transcription factor RXR and retinoic acid receptor (RAR), were significantly decreased only in T compared with Wt and P, although the oxidized polar metabolite of atRA, 4-keto-all-trans-retinoic-acid (4-keto-RA) was significantly decreased in both P and T compared with Wt. To our surprise, the functions of RXRα were significantly blocked only in T compared with Wt and P. Namely, the total protein levels of RXRα were significantly reduced and the phosphorylation levels of RXRα were significantly increased only in T compared with Wt and P. Treatment of H-ras12V transgenic mice at 5-week

PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

Science.gov (United States)

Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

2012-08-01

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells

Directory of Open Access Journals (Sweden)

Jesse E. Jun

2013-09-01

Full Text Available The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and SOS-family GEFs.Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood.One large group of biomolecules critically involved in the control of Ras-GEFs´functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells.

Evolutionary analyses of entire genomes do not support the association of mtDNA mutations with Ras/MAPK pathway syndromes.

Directory of Open Access Journals (Sweden)

Alberto Gómez-Carballa

Full Text Available BACKGROUND: There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS and related disorders (such as LEOPARD, neurofibromatosis type 1, although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM, which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45, most of them classified as NS patients (n = 42. METHODS/PRINCIPAL FINDINGS: The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg patterns of a typical Iberian dataset (including hgs H, T, J, and U. Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5 are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. CONCLUSIONS/SIGNIFICANCE: As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS.

Evolutionary Analyses of Entire Genomes Do Not Support the Association of mtDNA Mutations with Ras/MAPK Pathway Syndromes

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Cerezo, María; Balboa, Emilia; Heredia, Claudia; Castro-Feijóo, Lidia; Rica, Itxaso; Barreiro, Jesús; Eirís, Jesús; Cabanas, Paloma; Martínez-Soto, Isabel; Fernández-Toral, Joaquín; Castro-Gago, Manuel; Pombo, Manuel; Carracedo, Ángel; Barros, Francisco

2011-01-01

Background There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS) and related disorders (such as LEOPARD, neurofibromatosis type 1), although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA) genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM), which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45), most of them classified as NS patients (n = 42). Methods/Principal Findings The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg) patterns of a typical Iberian dataset (including hgs H, T, J, and U). Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5) are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. Conclusions/Significance As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS. PMID:21526175

Exploiting the bad eating habits of Ras-driven cancers.

Science.gov (United States)

White, Eileen

2013-10-01

Oncogenic Ras promotes glucose fermentation and glutamine use to supply central carbon metabolism, but how and why have only emerged recently. Ras-mediated metabolic reprogramming generates building blocks for growth and promotes antioxidant defense. To fuel metabolic pathways, Ras scavenges extracellular proteins and lipids. To bolster metabolism and mitigate stress, Ras activates cellular self-cannibalization and recycling of proteins and organelles by autophagy. Targeting these distinct features of Ras-driven cancers provides novel approaches to cancer therapy.

Association of Sphingosine-1-phosphate (S1P)/S1P Receptor-1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma.

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Rodriguez, A M; Graef, A J; LeVine, D N; Cohen, I R; Modiano, J F; Kim, J-H

2015-01-01

Sphingosine-1-phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs. S1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA). Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used. This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT-PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high-performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca(2+) mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining. Canine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1 , decreased S1P1 protein expression and induced apoptosis of HSA cells. S1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

EphB4 promotes or suppresses Ras/MEK/ERK pathway in a context-dependent manner: Implications for EphB4 as a cancer target.

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Xiao, Zhan; Carrasco, Rosa; Kinneer, Krista; Sabol, Darrin; Jallal, Bahija; Coats, Steve; Tice, David A

2012-06-01

EphB4 is a member of the Eph receptor tyrosine kinase family shown to act in neuronal guidance and mediate venal/arterial separation. In contrast to these more established roles, EphB4's function in cancer is much less clear. Here we illustrate both tumor promoting as well as suppressing roles of EphB4, by showing that its activation resulted in inhibition of the Ras/ERK pathway in endothelial cells but activation of the same pathway in MCF-7 breast cancer cells. This was true if EphB4 was stimulated with EphrinB2, its natural ligand, or an agonistic monoclonal antibody for EphB4. Correspondingly, EphB4 activation stimulated MCF7 growth while inhibiting HUVEC cell proliferation. The reason for these dramatic differences is due to functional coupling of EphB4 to different downstream effectors. Reduction of p120 RasGAP in HUVEC cells attenuated the inhibitory effect of EphB4 activation on the ERK pathway, whereas knockdown of PP2A in MCF7 cells attenuated EphB4 activation of the ERK pathway. This represents the first time a functional coupling between Eph receptor and PP2A has been demonstrated leading to activation of an oncogenic pathway. Our study illustrates the caveats and potential challenges of targeting EphB4 for cancer therapy due to the conflicting effects on cancer cell and endothelial cell compartments.

Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras

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Acin, Sergio; Li, Zhongyou; Mejia, Olga; Roop, Dennis R; El-Naggar, Adel K; Caulin, Carlos

2015-01-01

Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53R172H, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53R172H. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53R172H. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53. PMID:21952947

Ras Activity Oscillates in the Mouse Suprachiasmatic Nucleus and Modulates Circadian Clock Dynamics.

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Serchov, Tsvetan; Jilg, Antje; Wolf, Christian T; Radtke, Ina; Stehle, Jörg H; Heumann, Rolf

2016-04-01

Circadian rhythms, generated in the mouse suprachiasmatic nucleus (SCN), are synchronized to the environmental day-night changes by photic input. The activation of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) and cAMP response element-binding protein (CREB)-mediated transcription play a critical role in this photoentrainment. The small GTPase Ras is one of the major upstream regulators of the ERK1,2/CREB pathway. In contrast to the well-described role of Ras in structural and functional synaptic plasticity in the adult mouse brain, the physiological regulation of Ras by photic sensory input is yet unknown. Here, we describe for the first time a circadian rhythm of Ras activity in the mouse SCN. Using synRas transgenic mice, expressing constitutively activated V12-Ha-Ras selectively in neurons, we demonstrate that enhanced Ras activation causes shortening of the circadian period length. We found upregulated expression and decreased inhibitory phosphorylation of the circadian period length modulator, glycogen synthase kinase-3 beta (GSK3β), in the SCN of synRas mice. Conversely, downregulation of Ras activity by blocking its function with an antibody in oscillating cell cultures reduced protein levels and increased phosphorylation of GSK3β and lengthened the period of BMAL1 promoter-driven luciferase activity. Furthermore, enhanced Ras activity in synRas mice resulted in a potentiation of light-induced phase delays at early subjective night, and increased photic induction of pERK1,2/pCREB and c-Fos. In contrast, at late subjective night, photic activation of Ras/ERK1,2/CREB in synRas mice was not sufficient to stimulate c-Fos protein expression and phase advance the clock. Taken together, our results demonstrate that Ras activity fine tunes the period length and modulates photoentrainment of the circadian clock.

Cyclophilin B supports Myc and mutant p53-dependent survival of glioblastoma multiforme cells.

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Choi, Jae Won; Schroeder, Mark A; Sarkaria, Jann N; Bram, Richard J

2014-01-15

Glioblastoma multiforme is an aggressive, treatment-refractory type of brain tumor for which effective therapeutic targets remain important to identify. Here, we report that cyclophilin B (CypB), a prolyl isomerase residing in the endoplasmic reticulum (ER), provides an essential survival signal in glioblastoma multiforme cells. Analysis of gene expression databases revealed that CypB is upregulated in many cases of malignant glioma. We found that suppression of CypB reduced cell proliferation and survival in human glioblastoma multiforme cells in vitro and in vivo. We also found that treatment with small molecule inhibitors of cyclophilins, including the approved drug cyclosporine, greatly reduced the viability of glioblastoma multiforme cells. Mechanistically, depletion or pharmacologic inhibition of CypB caused hyperactivation of the oncogenic RAS-mitogen-activated protein kinase pathway, induction of cellular senescence signals, and death resulting from loss of MYC, mutant p53, Chk1, and Janus-activated kinase/STAT3 signaling. Elevated reactive oxygen species, ER expansion, and abnormal unfolded protein responses in CypB-depleted glioblastoma multiforme cells indicated that CypB alleviates oxidative and ER stresses and coordinates stress adaptation responses. Enhanced cell survival and sustained expression of multiple oncogenic proteins downstream of CypB may thus contribute to the poor outcome of glioblastoma multiforme tumors. Our findings link chaperone-mediated protein folding in the ER to mechanisms underlying oncogenic transformation, and they make CypB an attractive and immediately targetable molecule for glioblastoma multiforme therapy.

Dietary turmeric modulates DMBA-induced p21ras, MAP kinases and AP-1/NF-κB pathway to alter cellular responses during hamster buccal pouch carcinogenesis

International Nuclear Information System (INIS)

Garg, Rachana; Ingle, Arvind; Maru, Girish

2008-01-01

The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12 weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished the DMBA-induced mRNA expression of proto-oncogenes (c-jun, c-fos) and NF-κB, leading to decreased protein levels and in further attenuation of DMBA-induced AP-1/NF-κB DNA-binding in the buccal pouch nuclear extracts. Besides, buccal pouch of hamsters receiving turmeric diet showed significant alterations in DMBA-induced effects: (a) decrease in cell proliferation (diminished PCNA and Bcl2 expression), (b) enhanced apoptosis (increased expression of Bax, caspase-3 and apoptotic index), (c) decrease in inflammation (levels of Cox-2, the downstream target of AP-1/NF-κB, and PGE2) and (d) aberrant expression of differentiation markers, the cytokeratins (1, 5, 8, and 18). Together, the protective effects of dietary turmeric converge on augmenting apoptosis of the initiated cells and decreasing cell proliferation in DMBA-treated animals, which in turn, is reflected in decreased tumor burden, multiplicity and enhanced latency period. Some of these biomarkers are likely to be helpful in monitoring clinical trials and evaluating drug effect measurements

Genetic and Molecular Analysis of Suppressors of Ras Mutations

National Research Council Canada - National Science Library

Eastburn, Dennis

2000-01-01

.... The study of Caenorhabditis elegans and other model systems has demonstrated that Ras is part of a conserved Ras/MAPK signaling pathway involved in many aspects of development and cell regulation. The C...

Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation

Science.gov (United States)

Bettoun, Audrey; Surdez, Didier; Vallerand, David; Gundogdu, Ramazan; Sharif, Ahmad A.D.; Gomez, Marta; Cascone, Ilaria; Meunier, Brigitte; White, Michael A.; Codogno, Patrice; Parrini, Maria Carla; Camonis, Jacques H.; Hergovich, Alexander

2016-01-01

Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells. PMID:27283898

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

Science.gov (United States)

Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

2006-05-01

To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

Survival-related profile, pathways, and transcription factors in ovarian cancer.

Directory of Open Access Journals (Sweden)

Anne P G Crijns

2009-02-01

versus 41 mo, respectively; permutation p-value of log-rank statistic = 0.015 and maintained its independent prognostic value in multivariate analysis. Genes that composed the overall survival profile were also able to discriminate between the two risk groups in the independent dataset. In our dataset 17/167 pathways and 13/111 transcription factors were associated with overall survival, of which 16 and 12, respectively, were confirmed in the independent dataset. CONCLUSIONS: Our study provides new clues to genes, pathways, and transcription factors that contribute to the clinical outcome of serous ovarian cancer and might be exploited in designing new treatment strategies.

Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway

International Nuclear Information System (INIS)

Han, Seula; Woo, Jong Kyu; Jung, Yuchae; Jeong, Dawoon; Kang, Minsook; Yoo, Young-Ji; Lee, Hani; Oh, Seung Hyun; Ryu, Jae-Ha; Kim, Woo-Young

2016-01-01

In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. - Highlights: • Evodiamine selectively kills breast cancer stem like cells at G1 phase. • Evodiamine utilizes different mechanism of cell cycle modulation in CSLC and in bulk cancer cells. • Evodiamine activate the p53, p21 and Rb pathway.

Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway

Energy Technology Data Exchange (ETDEWEB)

Han, Seula [The Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul (Korea, Republic of); Woo, Jong Kyu [College of Pharmacy, Gachon University, Incheon (Korea, Republic of); Jung, Yuchae; Jeong, Dawoon; Kang, Minsook; Yoo, Young-Ji; Lee, Hani [The Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul (Korea, Republic of); Oh, Seung Hyun [College of Pharmacy, Gachon University, Incheon (Korea, Republic of); Ryu, Jae-Ha [The Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul (Korea, Republic of); Kim, Woo-Young, E-mail: wykim@sookmyung.ac.kr [The Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul (Korea, Republic of)

2016-01-22

In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. - Highlights: • Evodiamine selectively kills breast cancer stem like cells at G1 phase. • Evodiamine utilizes different mechanism of cell cycle modulation in CSLC and in bulk cancer cells. • Evodiamine activate the p53, p21 and Rb pathway.

Prostaglandin E2 blocks menadione-induced apoptosis through the Ras/Raf/Erk signaling pathway in promonocytic leukemia cell lines.

Science.gov (United States)

Yeo, Hyun-Seok; Shehzad, Adeeb; Lee, Young Sup

2012-04-01

Altered oxidative stress has long been observed in cancer cells, and this biochemical property of cancer cells represents a specific vulnerability that can be exploited for therapeutic benefit. The major role of an elevated oxidative stress for the efficacy of molecular targeted drugs is under investigation. Menadione is considered an attractive model for the study of oxidative stress, which can induce apoptosis in human leukemia HL-60 cell lines. Prostaglandin E(2) (PGE(2)) via its receptors not only promotes cell survival but also reverses apoptosis and promotes cancer progression. Here, we present evidence for the biological role of PGE(2) as a protective agent of oxidative stress-induced apoptosis in monocytic cells. Pretreatment of HL-60 cells with PGE(2) markedly ameliorated the menadione-induced apoptosis and inhibited the degradation of PARP and lamin B. The EP(2) receptor antagonist AH6809 abrogated the inhibitory effect of PGE(2), suggesting the role of the EP(2)/cAMP system. The PKA inhibitor H89 also reversed apoptosis and decreased the PKA activity that was elevated 10-fold by PGE(2). The treatment of HL-60 cells with NAC or zinc chloride showed a similar protective effect as with PGE(2) on menadione-treated cells. Furthermore, PGE(2) activated the Ras/Raf/MEK pathway, which in turn initiated ERK activation, and ultimately protected menadione-induced apoptosis. These results imply that PGE(2) via cell survival pathways may protect oxidative stress-induced apoptosis in monocytic cells. This study warrants further pre-clinical investigation as well as application towards leukemia clinics.

Characterization of sur-2, a Novel Ras-Mediated Signal Transduction Component in C. elegans

National Research Council Canada - National Science Library

DesJardins, Edward

1998-01-01

... (oncogenes). A subset of proto-oncogenes comprise the RAS signal transduction pathway. Vulval development in the nematode worm Caenorhabditis elegans is controlled by a RAS signal transduction pathway...

Effects of Germline Mutations in the Ras/MAPK Signaling Pathway on Adaptive Behavior: Cardiofaciocutaneous Syndrome and Noonan Syndrome

Science.gov (United States)

Pierpont, Elizabeth I.; Pierpont, Mary Ella; Mendelsohn, Nancy J.; Roberts, Amy E.; Tworog-Dube, Erica; Rauen, Katherine A.; Seidenberg, Mark S.

2011-01-01

Cardiofaciocutaneous syndrome (CFC) and Noonan syndrome (NS) are two phenotypically overlapping genetic disorders whose underlying molecular etiologies affect a common signaling pathway. Mutations in the BRAF, MEK1 and MEK2 genes cause most cases of CFC and mutations in PTPN11, SOS1, KRAS and RAF1 typically cause NS. Although both syndromes are associated with developmental delays of varying severity, the extent to which the behavioral profiles differ may shed light on the different roles these respective genes play in development of skills necessary for everyday functioning. In this study, profiles of adaptive behavior of individuals with CFC and NS who had confirmed pathogenic mutations in Ras/MAPK pathway genes were investigated. Patterns of strengths and weaknesses, age-related differences, and risk factors for difficulties in adaptive skills were assessed. Although genes acting more downstream in the Ras/MAPK pathway were associated with more difficulties in adaptive functioning than genes more upstream in the pathway, several inconsistencies highlight the wide spectrum of possible developmental courses in CFC and NS. Along with clinical and genetic factors, variables such as chronological age, gestational age at birth and parental education levels accounted for significant variance in adaptive skills. Results indicate that there is wide heterogeneity in adaptive ability in CFC and NS, but that these abilities are correlated to some extent with the specific disease-causing genes. PMID:20186801

Characterization of sur-2, a Novel Ras-Mediated Signal Transduction Component in C. elegans

National Research Council Canada - National Science Library

DesJardins, Edward

1999-01-01

... (oncogenes). A subset of proto-oncogenes comprise the RAS signal transduction pathway. Vulval development in the nematode worm Caenorhabditis elegans is controlled by a RAS signal transduction pathway. C...

RasGRP1, but not RasGRP3, is required for efficient thymic β-selection and ERK activation downstream of CXCR4.

Directory of Open Access Journals (Sweden)

Dominic P Golec

Full Text Available T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-CD8(- 'double negative' (DN thymocytes, pass through a checkpoint termed "β-selection" before maturing into CD4(+CD8(+ 'double positive' (DP thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ(+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

p53, erbB-2 and K-ras gene alterations are rare in spontaneous and plutonium-239-induced canine lung neoplasia

International Nuclear Information System (INIS)

Tierney, L.A.; Hahn, F.F.; Lechner, J.F.

1996-01-01

Inhalation of high-linear energy transfer radiation in the form of radon progeny is a suspected cause of human lung cancer. To gain insight into the types of genetic derangements caused by this type of radiation, lung tumors from beagle dogs exposed to 239 PuO 2 and those arising in animals with no known carcinogen exposure were examined for evidence of aberrations in genes known to be altered in lung tumors. Altered expression of the p53 tumor suppressor gene and proto-oncogene erbB-2 proteins (p185 erbB2 ) was evaluated by immunohistochemical analysis of 117 tumors representing different histological types in exposed (n = 80) and unexposed (n = 37) animals. Twenty-eight tumors were analyzed for K-ras proto-oncogene mutations by polymerase chain reaction amplification and direct sequencing. Fourteen percent (16/116) of all lung neoplasms showed elevated nuclear accumulation of p53 protein. Regardless of exposure history, adenosquamous and squamous cell cancers comprised 94% of all tumors with p53 abnormalities. Eighteen percent (21/117) of all tumors had evidence of erbB-2 protein overexpression. K-ras mutations were not detected in codons 12, 13 or 61 of tumors from unexposed (n = 9) or plutonium-exposed dogs (n = 19). These data indicate that p53 and K-ras gene abnormalities as a result of missense mutation are infrequent events in spontaneous and 239 PuO 2 -induced lung neoplasia in this colony of beagle dogs. Alternative mechanisms of gene alteration may be involved in canine pulmonary carcinogenesis. 45 refs., 3 figs., 2 tabs

Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Fujiyama, A.; Tamanoi, F.

1986-01-01

The authors demonstrate the pathway for the biosynthesis of RAS1 and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes. The primary translation products of these genes are detected in a soluble fraction. Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO 4 /polyacrylamide gel. These processed proteins are further modified by fatty acid acylation, which is detected by [ 3 H]palmitic acid labeling. The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process. The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond. The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins. Fraction of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane

The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human renal cell carcinoma cell growth.

Science.gov (United States)

Su, Ying; Zhao, An; Cheng, Guoping; Xu, Jingjing; Ji, Enming; Sun, Wenyong

2017-07-04

Renal cell carcinoma (RCC) is the highest mortality rate of the genitourinary cancers, and the treatment options are very limited. Thus, identification of molecular mechanisms underlying RCC tumorigenesis, is critical for identifying biomarkers for RCC diagnosis and prognosis. To validate whether the IGF-I/JAK2-STAT3/miR-21 signaling pathway is associated with human RCC cell growth. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels, respectively. The MTT assay was performed to determine cell survival rate. The Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. We employed RCC tissues and cell lines (A498; ACHN; Caki-1; Caki-2 and 786-O) in the study. IGF-I, and its inhibitor (NT-157) were administrated to detect the effects of IGF-I on the expression of miR-21 and p-JAK2. JAK2 inhibitor (AG490), and si-STAT3 were used to detect the effects of JAK2/STAT3 signaling pathway on the expression of miR-21. In our study, we firstly showed that the expression levels of IGF-I and miR-21 were up-regulated in RCC tissues and cell lines. After exogenous IGF-I treatment, the expression levels of miR-21, p-IGF-IR and p-JAK2 were significantly increased, whereas NT-157 treatment showed the reversed results. Further study indicated that JAK2 inhibitor or si-STAT3 significantly reversed the IGF-I-induced miR-21 expression level. Finally, we found that IGF-I treatment significantly prompted human RCC cell survival and inhibited cell apoptosis, and NT-157 treatment showed the reversed results. The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human RCC cell growth.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

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Vanli, Güliz; Peltzer, Nieves; Dubuis, Gilles; Widmann, Christian

2014-12-01

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N. Copyright © 2014 Elsevier Inc. All rights reserved.

Oncogenic Activation of Fibroblast Growth Factor Receptor-3 and RAS Genes as Non-Overlapping Mutual Exclusive Events in Urinary Bladder Cancer.

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Pandith, Arshad A; Hussain, Aashaq; Khan, Mosin S; Shah, Zafar A; Wani, M Saleem; Siddiqi, Mushtaq A

2016-01-01

Urinary bladder cancer is a common malignancy in the West and ranks as the 7th most common cancer in our region of Kashmir, India. FGFR3 mutations are frequent in superficial urothelial carcinoma (UC) differing from the RAS gene mutational pattern. The aim of this study was to analyze the frequency and association of FGFR3 and RAS gene mutations in UC cases. Paired tumor and adjacent normal tissue specimens of 65 consecutive UC patients were examined. DNA preparations were evaluated for the occurrence of FGFR3 and RAS gene mutations by PCR-SCCP and DNA sequencing. Somatic point mutations of FGFR3 were identified in 32.3% (21 of 65). The pattern and distribution were significantly associated with low grade/stage (<0.05). The overall mutations in exon 1 and 2 in all the forms of RAS genes aggregated to 21.5% and showed no association with any clinic-pathological parameters. In total, 53.8% (35 of 65) of the tumors studied had mutations in either a RAS or FGFR3 gene, but these were totally mutually exclusive in and none of the samples showed both the mutational events in mutually exclusive RAS and FGFR3. We conclude that RAS and FGFR3 mutations in UC are mutually exclusive and non-overlapping events which reflect activation of oncogenic pathways through different elements.

Ras pathway activation in gliomas: a strategic target for intranasal administration of perillyl alcohol

International Nuclear Information System (INIS)

Fonseca, C. O.; Linden, R.; Futuro, D.; Gattass, C.R.; Quirico-Santos, T.

2008-01-01

Targeted therapy directed at specific molecular alterations is already creating a shift in the treatment of cancer patients. Malignant gliomas commonly overexpress the oncogenes EGFR and PDGFR and contain mutations and deletions of the tumor suppressor genes PTEN and TP53. Some of these alterations lead to activation of the P13K/Akt and Ras/MAPK pathways, which provide targets for therapy. Perillyl alcohol (POH), the isoprenoid of greatest clinical interest, was initially considered to inhibit farnesyl protein transferase. Follow-up studies revealed that POH suppresses the synthesis of small G proteins, including Ras. Intranasal delivery allows drugs that do not cross the blood-brain barrier to enter the central nervous system. Moreover, it eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects. Applying this method, a phase I/II clinical trial of POH was performed in patients with relapsed malignant gliomas after standard treatment: surgery, radiotherapy, and chemotherapy. POH was administrated in a concentration of 0.3% volume/volume (55 mg) four times daily in an interrupted administration schedule. The objective was to evaluate toxicity and progression-free survival (PFS) after six months of treatment. The cohort consisted of 37 patients, including 29 with glioblastoma multiforme (GBM), 5 with grade III astrocytoma (AA), and 3 with anaplastic oligodendroglioma (AO). Neurological examination and suitable image analysis (computed tomography (CT), magnetic resonance imaging (MRI)) established disease progression. Complete response was defined as neurological stability or improvement of conditions, disappearance of CT/MRI tumor image, and corticosteroid withdraw; partial response (PR) as .50% reduction of CT/MRI tumor image, neurological stability, or improvement of conditions and corticosteroid requirement; progressive course (PC) as .25% increase in CT/MRI tumor image or the appearance of a new lesion; and stable disease as a

BMP suppresses PTEN expression via RAS/ERK signaling.

Science.gov (United States)

Beck, Stayce E; Carethers, John M

2007-08-01

Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

Science.gov (United States)

Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

2005-06-06

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

International Nuclear Information System (INIS)

Dominguez, J.M.; Lanoix, J.; Paiement, J.

1991-01-01

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha- 32 P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha- 32 P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations

The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Toward a Therapeutic Approach

OpenAIRE

Korf, Bruce; Ahmadian, Reza; Allanson, Judith; Aoki, Yoko; Bakker, Annette; Wright, Emma Burkitt; Denger, Brian; Elgersma, Ype; Gelb, Bruce D.; Gripp, Karen W.; Kerr, Bronwyn; Kontaridis, Maria; Lazaro, Conxi; Linardic, Corinne; Lozano, Reymundo

2015-01-01

"The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach" was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regard...

The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

International Nuclear Information System (INIS)

Plowman, Sarah J.; Arends, Mark J.; Brownstein, David G.; Luo Feijun; Devenney, Paul S.; Rose, Lorraine; Ritchie, Ann-Marie; Berry, Rachel L.; Harrison, David J.; Hooper, Martin L.; Patek, Charles E.

2006-01-01

Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras tmΔ4A/tmΔ4A (exon 4A knock-out) ES cells which express K-ras4B only and K-ras -/- (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras tmΔ4A/tmΔ4A mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras tmΔ4A/tmΔ4A ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras tmΔ4A/tmΔ4A and K-ras -/- ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras -/- cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras tmΔ4A/tmΔ4A ES cells were more resistant to etoposide-induced apoptosis than K-ras -/- cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice

BRAF and RAS oncogenes regulate Rho GTPase pathways to mediate migration and invasion properties in human colon cancer cells: a comparative study

Directory of Open Access Journals (Sweden)

Shirasawa Senji

2011-09-01

Full Text Available Abstract Background Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A, Rac1 (Ras-related C3 botulinum toxin substrate 1 and Cdc42 (cell division cycle 42 in cancer progression induced by each of the three oncogenes is described. Methods Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities. Results Evidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT were the main characteristics rendered by HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFβ-1 pathway to provide cells with additional transforming

Oncogenic N-Ras Stimulates SRF-Mediated Transactivation via H3 Acetylation at Lysine 9

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Sun-Ju Yi

2018-01-01

Full Text Available Signal transduction pathways regulate the gene expression by altering chromatin dynamics in response to mitogens. Ras proteins are key regulators linking extracellular stimuli to a diverse range of biological responses associated with gene regulation. In mammals, the three ras genes encode four Ras protein isoforms: H-Ras, K-Ras4A, K-Ras4B, and N-Ras. Although emerging evidence suggests that Ras isoforms differentially regulate gene expressions and are functionally nonredundant, the mechanisms underlying Ras specificity and Ras signaling effects on gene expression remain unclear. Here, we show that oncogenic N-Ras acts as the most potent regulator of SRF-, NF-κB-, and AP-1-dependent transcription. N-Ras-RGL2 axis is a distinct signaling pathway for SRF target gene expression such as Egr1 and JunB, as RGL2 Ras binding domain (RBD significantly impaired oncogenic N-Ras-induced SRE activation. By monitoring the effect of Ras isoforms upon the change of global histone modifications in oncogenic Ras-overexpressed cells, we discovered that oncogenic N-Ras elevates H3K9ac/H3K23ac levels globally in the chromatin context. Importantly, chromatin immunoprecipitation (ChIP assays revealed that H3K9ac is significantly enriched at the promoter and coding regions of Egr1 and JunB. Collectively, our findings define an undocumented role of N-Ras in modulating of H3 acetylation and in gene regulation.

The Yeast Saccharomyces cerevisiae as a Model for Understanding RAS Proteins and Their Role in Human Tumorigenesis

Science.gov (United States)

Cazzanelli, Giulia; Francisco, Rita; Azevedo, Luísa; Carvalho, Patrícia Dias; Almeida, Ana; Côrte-Real, Manuela; Oliveira, Maria José; Lucas, Cândida; Sousa, Maria João

2018-01-01

The exploitation of the yeast Saccharomyces cerevisiae as a biological model for the investigation of complex molecular processes conserved in multicellular organisms, such as humans, has allowed fundamental biological discoveries. When comparing yeast and human proteins, it is clear that both amino acid sequences and protein functions are often very well conserved. One example of the high degree of conservation between human and yeast proteins is highlighted by the members of the RAS family. Indeed, the study of the signaling pathways regulated by RAS in yeast cells led to the discovery of properties that were often found interchangeable with RAS proto-oncogenes in human pathways, and vice versa. In this work, we performed an updated critical literature review on human and yeast RAS pathways, specifically highlighting the similarities and differences between them. Moreover, we emphasized the contribution of studying yeast RAS pathways for the understanding of human RAS and how this model organism can contribute to unveil the roles of RAS oncoproteins in the regulation of mechanisms important in the tumorigenic process, like autophagy. PMID:29463063

RSK is a principal effector of the RAS-ERK pathway for eliciting a coordinate promotile/invasive gene program and phenotype in epithelial cells

DEFF Research Database (Denmark)

Doehn, Ulrik; Hauge, Camilla; Frank, Scott R

2009-01-01

The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during development, tissue regeneration, and carcinoma progression, often via promoting the epithelial-mesenchymal transition (EMT). Many mechanisms by which ERK exerts this control remain...... elusive. We demonstrate that the ERK-activated kinase RSK is necessary to induce mesenchymal motility and invasive capacities in nontransformed epithelial and carcinoma cells. RSK is sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK...... to stimulate motility and invasion. These findings uncover a mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanate multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties....

miR-654-5p Targets GRAP to Promote Proliferation, Metastasis, and Chemoresistance of Oral Squamous Cell Carcinoma Through Ras/MAPK Signaling.

Science.gov (United States)

Lu, Meng; Wang, Chengyong; Chen, Weihui; Mao, Chuanqing; Wang, Jin

2018-04-01

Oral squamous cell carcinoma (OSCC) is characterized by rapid local migration and invasion. This study was aimed at clarifying the effect of miR-654-5p on progression of OSCC. miR-654-5p promoted proliferation, metastasis, and chemoresistance of OSCC in vitro and in vivo. Consistently, miR-654-5p was upregulated in late-stage OSCC and was correlated with poor prognosis of OSCC patients. Furthermore, miR-654-5p was mechanistically verified to target Grb-2-related adaptor protein (GRAP), accompanied by the activation of Ras/MAPK signaling and the facilitation of epithelial-mesenchymal transition in OSCC cells. GRAP was downregulated in T1-2 stage versus T3-4 stage head and neck squamous cell carcinoma (HNSC) and was negatively correlated with tumor-node-metastases (TNM) stage in HNSC patients based on The Cancer Genome Atlas (TCGA) analysis. In addition, GRAP was positively correlated with good prognosis in HNSC patients. Our findings suggest that the miR-654-5p/GRAP/Ras/Erk signaling pathway in OSCC cells might contribute to the underlying mechanism through which miR-654-5p participates in the regulation of OSCC progression. miR-654-5p, as a potential biomarker for the clinical diagnosis and prognosis of OSCC, may be an effective anticancer target for the treatment of OSCC.

ACE variants interact with the RAS pathway to confer risk and protection against type 2 diabetic nephropathy

DEFF Research Database (Denmark)

Ahluwalia, Tarun Veer Singh; Ahuja, Monica; Rai, Taranjit Singh

2009-01-01

Genetic predisposition has been proposed to be a major determinant in the development of renal complications of diabetes. Among candidate genes examined for susceptibility to diabetic nephropathy, angiotensin-converting enzyme (ACE) gene has been found to be associated with pathogenesis......, in the present study, we evaluated the association of ACE haplotypes and the interactions of ACE, angiotensinogen (AGT), and angiotensin II receptor type I (AGTR1) gene polymorphisms with DNP in Asian Indians. We genotyped seven variants of the RAS pathway genes (ACE, AGT, and AGTR1) in type 2 diabetic cohorts...... and progression of diabetic nephropathy. However, the role of other renin-angiotensin system (RAS) polymorphisms and their possible interactions with different ACE I/D genotypes are less clearly defined. Recent studies also show that ACE haplotypes may be better predictors to disease susceptibility. Thus...

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

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Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P ras4B can enhance the expression of ERalpha and

Ras Signaling Regulates Stem Cells and Amelogenesis in the Mouse Incisor.

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Zheng, X; Goodwin, A F; Tian, H; Jheon, A H; Klein, O D

2017-11-01

The role of Ras signaling during tooth development is poorly understood. Ras proteins-which are activated by many upstream pathways, including receptor tyrosine kinase cascades-signal through multiple effectors, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. Here, we utilized the mouse incisor as a model to study how the MAPK and PI3K pathways regulate dental epithelial stem cells and amelogenesis. The rodent incisor-which grows continuously throughout the life of the animal due to the presence of epithelial and mesenchymal stem cells-provides a model for the study of ectodermal organ renewal and regeneration. Utilizing models of Ras dysregulation as well as inhibitors of the MAPK and PI3K pathways, we found that MAPK and PI3K regulate dental epithelial stem cell activity, transit-amplifying cell proliferation, and enamel formation in the mouse incisor.

Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress.

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Stacey L Lehman

2015-06-01

Full Text Available Multiple transcripts encode for the cell cycle inhibitor p21(Cip1. These transcripts produce identical proteins but differ in their 5' untranslated regions (UTRs. Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through (35S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5' upstream open reading frames (uORFs through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress.

Black Rice Anthocyanins Suppress Metastasis of Breast Cancer Cells by Targeting RAS/RAF/MAPK Pathway.

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Chen, Xiang-Yan; Zhou, Jie; Luo, Li-Ping; Han, Bin; Li, Fei; Chen, Jing-Yao; Zhu, Yan-Feng; Chen, Wei; Yu, Xiao-Ping

2015-01-01

Overexpression of human epidermal growth factor receptor 2 (HER2) drives the biology of 30% of breast cancer cases. As a transducer of HER2 signaling, RAS/RAF/MAPK pathway plays a pivotal role in the development of breast cancer. In this study, we examined the molecular mechanisms underlying the chemopreventive effects of black rice anthocyanins (BRACs) extract and identified their molecular targets in HER2(+) breast cancer cells. Treatment of MDA-MB-453 cells (HER2(+)) with BRACs inhibited cell migration and invasion, suppressed the activation of mitogen-activated protein kinase kinase kinase (RAF), mitogen-activated protein kinase kinase (MEK), and c-Jun N-terminal kinase (JNK), and downregulated the secretion of matrix metalloproteinase 2 (MMP2) and MMP9. BRACs also weakened the interactions of HER2 with RAF, MEK, and JNK proteins, respectively, and decreased the mRNA expression of raf, mek, and jnk. Further, we found combined treatment with BRACs and RAF, MEK, or JNK inhibitors could enhance the antimetastatic activity, compared with that of each treatment. Transient transfection with small interfering RNAs (siRNAs) specific for raf, mek, and jnk inhibited their mRNA expression in MDA-MB-453 cells. Moreover, cotreatment with BRACs and siRNA induces a more remarkable inhibitory effect than that by either substance alone. In summary, our study suggested that BRACs suppress metastasis in breast cancer cells by targeting the RAS/RAF/MAPK pathway.

Acquired resistance to cetuximab is associated with the overexpression of Ras family members and the loss of radiosensitization in head and neck cancer cells

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Saki, Mohammad; Toulany, Mahmoud; Rodemann, H. Peter

2013-01-01

Purpose: Cetuximab in combination with radiation therapy is used to treat patients with head and neck squamous cell carcinoma (HNSCC). In the present study, the mechanism of acquired resistance to cetuximab in HNSCC cells was investigated in vitro. Material and methods: The HNSCC cell lines UT5 and SAS and UT5 cells with acquired resistance to cetuximab (UT5R9) were used. The radiotoxicity potentials of cetuximab and inhibitors of PI3K, MAPK and farnesylation were tested using a clonogenic survival assay. Western blotting was used to evaluate protein expression. The levels of EGFR ligands were detected by ELISA. Results: Cetuximab inhibited proliferation and induced radiosensitization in UT5 cells but not in SAS cells. In comparison with UT5 cells, cetuximab-resistant SAS cells markedly overexpressed the K-Ras, H-Ras and N-Ras proteins, as detected by Western blotting. Resistance in UT5R9 cells was associated with the overexpression of the K-Ras, H-Ras and N-Ras proteins as well as an increase in the autocrine production of the EGFR ligands amphiregulin and transforming growth factor α (TGFα). UT5R9 cells were significantly more radioresistant than UT5 cells. Radioresistant UT5R9 cells were not radiosensitized by cetuximab, but knocking down H-RAS and N-RAS with siRNA and targeting Ras farnesylation using the farnesyltransferase inhibitor lonafarnib induced radiosensitization in these cells. Targeting PI3K and MEK revealed that the activation of the PI3K/Akt pathway but not the MAPK/ERK pathway is associated with radioresistance in UT5R9 cells. Conclusion: Targeting Ras and PI3K activity improves the outcome of irradiation in cetuximab-resistant HNSCC cell lines in vitro

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

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Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd

PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers.

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Filip Janku

Full Text Available Oncogenic mutations of PIK3CA, RAS (KRAS, NRAS, and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.Tumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS, and BRAF mutations using polymerase chain reaction-based DNA sequencing.PIK3CA mutations were found in 54 (11% of 504 patients tested; KRAS in 69 (19% of 367; NRAS in 19 (8% of 225; and BRAF in 31 (9% of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%, uterine (7/28, 25%, breast (6/29, 21%, and colorectal cancers (18/105, 17%; KRAS in pancreatic (5/9, 56%, colorectal (49/97, 51%, and uterine cancers (3/20, 15%; NRAS in melanoma (12/40, 30%, and uterine cancer (2/11, 18%; BRAF in melanoma (23/52, 44%, and colorectal cancer (5/88, 6%. Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wtPIK3CA (p = 0.001. In total, RAS (KRAS, NRAS or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001. PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001 and in 20% of patients with RAS (KRAS, NRAS or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS or wtBRAF (p = 0.001.PIK3CA, RAS (KRAS, NRAS, and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS and BRAF mutations.

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

International Nuclear Information System (INIS)

Yu, Zhendong; Wang, Hao; Zhang, Libin; Tang, Aifa; Zhai, Qinna; Wen, Jianxiang; Yao, Li; Li, Pengfei

2009-01-01

CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

Energy Technology Data Exchange (ETDEWEB)

Yu, Zhendong, E-mail: zdyu@hotmail.com [Department of Clinical laboratory, Peking University Shenzhen Hospital, Guangdong (China); Wang, Hao [Department of pathology, The Chinese University of Hong Kong, Hong Kong (China); Zhang, Libin; Tang, Aifa; Zhai, Qinna; Wen, Jianxiang; Yao, Li [Department of Clinical laboratory, Peking University Shenzhen Hospital, Guangdong (China); Li, Pengfei, E-mail: lipengfei@cuhk.edu.hk [Department of pathology, The Chinese University of Hong Kong, Hong Kong (China)

2009-09-04

CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.

H-RAS, K-RAS, and N-RAS gene activation in human bladder cancers.

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Przybojewska, B; Jagiello, A; Jalmuzna, P

2000-08-01

Bladder cancer is one of the leading causes of cancer death in most developed countries. In this work, 19 bladder cancer specimens, along with their infiltrations of the urinary bladder wall from the same patients, were examined for the presence of H-RAS, K-RAS, and N-RAS activation using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The H-RAS activation was found in 15 (about 84%) of the 19 bladder cancers studied. The same results were obtained in the infiltrating urinary bladder wall samples. N-RAS gene mutations were observed in all cases (except 1) in which H-RAS gene mutations were detected. The results suggest a strong relationship between H-RAS and N-RAS gene activation in bladder cancer. Changes in the K-RAS gene in bladder cancers seem to be a rare event; this is in agreement with findings of other authors. We found activation of the gene in one specimen of bladder cancer and its infiltration of the urinary bladder wall in the same patient.

Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

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Wei-Ru Huang

Full Text Available Avian reovirus (ARV protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128 of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

MiR-146b-5p overexpression attenuates stemness and radioresistance of glioma stem cells by targeting HuR/lincRNA-p21/β-catenin pathway

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Yang, Wei; Yu, Hongquan; Shen, Yueming; Liu, Yingying; Yang, Zhanshan; Sun, Ting

2016-01-01

A stem-like subpopulation existed in GBM cells, called glioma stem cells (GSCs), might contribute to cancer invasion, angiogenesis, immune evasion, and therapeutic resistance, providing a rationale to eliminate GSCs population and their supporting niche for successful GBM treatment. LincRNA-p21, a novel regulator of cell proliferation, apoptosis and DNA damage response, is found to be downregulated in several types of tumor. However, little is known about the role of lincRNA-p21 in stemness and radioresistance of GSCs and its regulating mechanisms. In this study, we found that lincRNA-p21 negatively regulated the expression and activity of β-catenin in GSCs. Downregulation of lincRNA-p21 in GSCs was resulted from upregulation of Hu antigen R (HuR) expression caused by miR-146b-5p downregulation. MiR-146b-5p overexpression increased apoptosis and radiosensitivity, decreased cell viability, neurosphere formation capacity and stem cell marker expression, and induced differentiation in GSCs. Moreover, knock-down lincRNA-p21 or HuR and β-catenin overexpression could rescue the phenotypic changes resulted from miR-146b-5p overexpression in GSCs. These findings suggest that targeting the miR-146b-5p/HuR/lincRNA-p21/β-catenin signaling pathway may be valuable therapeutic strategies against glioma. PMID:27166258

NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

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Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

2018-05-02

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

Qingfei Xiaoyan Wan, a traditional Chinese medicine formula, ameliorates Pseudomonas aeruginosa–induced acute lung inflammation by regulation of PI3K/AKT and Ras/MAPK pathways

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Yuanyuan Hou

2016-05-01

Full Text Available Gram-negative pathogen–induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF, a traditional Chinese medicine (TCM formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosa–induced acute pneumonia in mice were evaluated. The mechanisms by which four typical anti-inflammatory ingredients from QF, arctigenin (ATG, cholic acid (CLA, chlorogenic acid (CGA and sinapic acid (SPA, regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6 and chemokines (IL-8 and RANTES, reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively.

Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

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Jennifer S. Liu

2012-11-01

Full Text Available Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

Expression of ras oncogene and major histocompatibility complex (MHC) antigen in carcinomas of the uterine cervix

International Nuclear Information System (INIS)

Cho, Kyung Ja; Jang, Ja June; Kim, Yong Dae; Ha, Chang Won; Koh, Jae Soo

1993-01-01

Consecutive 50 cases of squamous cell carcinomas of the uterine cervix diagnosed in 1992 were subjected to immunohistochemical study for ras oncogene product (p21) and MHC class II (DR) antigen using a microprobe immunostainer. Activated ras and aberrant DR expression were noted in 26 cases (52%) and 11 cases (22%) of cervical squamous cell carcinomas, respectively, without difference among histologic types. The reaction was mainly intracytoplasmic, with granular staining pattern and diffuse distribution. No direct histologic correlation between ras and DR expression was found. Four cases with HPV 16/18 DNA in superficial koilocytotic cells, revealed by in situ hybridization, showed various expression of ras and DR, and these 3 factors histologically did not seem to be affected one another. (Author)

Oncogenic RAS enables DNA damage- and p53-dependent differentiation of acute myeloid leukemia cells in response to chemotherapy.

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Mona Meyer

Full Text Available Acute myeloid leukemia (AML is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.

Fungal communication requires the MAK-2 pathway elements STE-20 and RAS-2, the NRC-1 adapter STE-50 and the MAP kinase scaffold HAM-5.

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Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

2014-11-01

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell-cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell-cell communication in fungi and higher eukaryotes.

MRAS: A Close but Understudied Member of the RAS Family.

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Young, Lucy C; Rodriguez-Viciana, Pablo

2018-01-08

MRAS is the closest relative to the classical RAS oncoproteins and shares most regulatory and effector interactions. However, it also has unique functions, including its ability to function as a phosphatase regulatory subunit when in complex with SHOC2 and protein phosphatase 1 (PP1). This phosphatase complex regulates a crucial step in the activation cycle of RAF kinases and provides a key coordinate input required for efficient ERK pathway activation and transformation by RAS. MRAS mutations rarely occur in cancer but deregulated expression may play a role in tumorigenesis in some settings. Activating mutations in MRAS (as well as SHOC2 and PP1) do occur in the RASopathy Noonan syndrome, underscoring a key role for MRAS within the RAS-ERK pathway. MRAS also has unique roles in cell migration and differentiation and has properties consistent with a key role in the regulation of cell polarity. Further investigations should shed light on what remains a relatively understudied RAS family member. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Prognostic implications of c-Ki-ras2 mutations in patients with advanced colorectal cancer treated with 5-fluorouracil and interferon: a study of the eastern cooperative oncology group (EST 2292)

Science.gov (United States)

Wadler, S; Bajaj, R; Neuberg, D; Agarwal, V; Haynes, H; Benson, A B

1997-01-01

Mutations in c-Ki-ras2 (ras) occur in about 40% of patients with colorectal cancers and occur early in the pathogenesis of this disease. To evaluate the prognostic value of mutations in ras, the Eastern Cooperative Oncology Group (ECOG) conducted a retrospective study (EST 2292) to determine the frequency of mutations in patients with advanced colorectal cancer, and to determine whether ras mutations were associated with altered response to therapy and survival. Patients were enrolled from four studies: P-Z289, an ECOG phase II trial of 5-fluorouracil (5-FU) and interferon (IFN) in patients with advanced colorectal cancer; P-Z991, an ECOG phase I trial of 5-FU and IFN in patients with advanced malignancies; and two trials from the Albert Einstein College of Medicine in patients with advanced colorectal cancer treated with 5-FU and either IFN-alpha or IFN-beta. All patients had advanced colorectal carcinoma and had sufficient histologic material available for analysis for the presence and type of ras, using polymerase chain reaction and dot-blot analysis with sets of probes sufficient to detect all the common mutations of ras at codons 12, 13, and 61. Seventy-two patients were enrolled in this trial. Mutations in ras were detected in 25 (35%), including 17 (23%) in codon 12, four (6%) in codon 13, and four (6%) in codon 61. There was no correlation between the presence of a ras mutation and age, sex, Dukes' stage, histology, or tumor markers. Thirty-one of 72 patients (43%) responded to therapy with 5-FU and IFN, and 10 of 31 responders (32%) and 15 of 41 nonresponders (37%) had mutations in ras. There was no difference in response rates or overall survival between the groups with and without ras mutations. It is unlikely that ras mutations will have significant prognostic value for either response to therapy or survival in patients with colorectal carcinomas treated with 5-FU and IFN.

Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains

DEFF Research Database (Denmark)

Willumsen, B M; Papageorge, A G; Hubbert, N

1985-01-01

or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences...... that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids...... that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor...

The p75NTR mediates a bifurcated signal transduction cascade through the NFκB and JNK pathways to inhibit cell survival

International Nuclear Information System (INIS)

Allen, Jeffrey; Khwaja, Fatima; Byers, Stephen; Djakiew, Daniel

2005-01-01

p75 NTR is most abundantly expressed in the nervous system, but is also widely expressed in many other organs and tissues where it primarily functions as a negative regulator of cell survival. In the prostate, p75 NTR functions as an inhibitory protein capable of slowing proliferation and inducing apoptosis. It has been shown that p75 NTR is expressed in the normal prostate, progressively lost from malignant tumor cells in vivo, and largely absent from prostate cancer cell lines derived from metastases. Although the role of p75 NTR in prostate cancer has been well established, the signal transduction pathway that mediates its inhibitory activity has only been partially elucidated. This study demonstrates that exogenous expression of p75 NTR down-regulates, in a dose-dependent manner, a bifurcated signaling cascade that results in reduced expression of potent transcription effectors. This two-arm signal transduction cascade was directly linked to the upstream receptor by using dominant-negative deletion constructs of p75 NTR that rescued tumor cells from p75 NTR -induced loss of survival and promotion of apoptosis. Furthermore, the dominant negatives rescued alterations in the levels of signal transduction intermediates. Conversely, the use of kinase-inactive intermediates that are downstream of the receptor further reduced expression of involved transcription effectors and reduced survival of the cells. These results provide a definitive link between the proximate p75 NTR and signal transduction intermediates leading to the transcription effectors NFκB and JNK, with associated growth suppression and induction of apoptosis

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

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Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-10-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

The Expression, Purification, and Characterization of a Ras Oncogene (Bras2) in Silkworm (Bombyx mori)

OpenAIRE

Lv, Zhengbing; Wang, Tao; Zhuang, Wenhua; Wang, Dan; Chen, Jian; Nie, Zuoming; Liu, Lili; Zhang, Wenping; Wang, Lisha; Wang, Deming; Wu, Xiangfu; Li, Jun; Qian, Lian; Zhang, Yaozhou

2013-01-01

The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412 bp. The open reading frame contains 603 bp, which encodes 200 amino acid residues. This recombinant BmBras2 protein was subsequently used as an antigen to raise a rabbit polyclonal antibody. Western blott...

Regulation of Hxt3 and Hxt7 turnover converges on the Vid30 complex and requires inactivation of the Ras/cAMP/PKA pathway in Saccharomyces cerevisiae.

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Chris Snowdon

Full Text Available Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c, an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.

The thrombopoietin/MPL/Bcl-xL pathway is essential for survival and self-renewal in human preleukemia induced by AML1-ETO

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Chou, Fu-Sheng; Griesinger, Andrea; Wunderlich, Mark; Lin, Shan; Link, Kevin A.; Shrestha, Mahesh; Goyama, Susumu; Mizukawa, Benjamin; Shen, Shuhong; Marcucci, Guido

2012-01-01

AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO–induced MPL. PMID:22337712

Activated platelet-derived growth factor β receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas.

Science.gov (United States)

Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe

2012-03-01

Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2). Copyright © 2011 Elsevier Ltd. All rights reserved.

Sub-chronic agmatine treatment modulates hippocampal neuroplasticity and cell survival signaling pathways in mice.

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Freitas, Andiara E; Bettio, Luis E B; Neis, Vivian B; Moretti, Morgana; Ribeiro, Camille M; Lopes, Mark W; Leal, Rodrigo B; Rodrigues, Ana Lúcia S

2014-11-01

Agmatine is an endogenous neuromodulator which, based on animal and human studies, is a putative novel antidepressant drug. In this study, we investigated the ability of sub-chronic (21 days) p.o. agmatine administration to produce an antidepressant-like effect in the tail suspension test and examined the hippocampal cell signaling pathways implicated in such an effect. Agmatine at doses of 0.01 and 0.1 mg/kg (p.o.) produced a significant antidepressant-like effect in the tail suspension test and no effect in the open-field test. Additionally, agmatine (0.001-0.1 mg/kg, p.o.) increased the phosphorylation of protein kinase A substrates (237-258% of control), protein kinase B/Akt (Ser(473)) (116-127% of control), glycogen synthase kinase-3β (Ser(9)) (110-113% of control), extracellular signal-regulated kinases 1/2 (119-137% and 121-138% of control, respectively) and cAMP response elements (Ser(133)) (127-152% of control), and brain-derived-neurotrophic factor (137-175% of control) immunocontent in a dose-dependent manner in the hippocampus. Agmatine (0.001-0.1 mg/kg, p.o.) also reduced the c-jun N-terminal kinase 1/2 phosphorylation (77-71% and 65-51% of control, respectively). Neither protein kinase C nor p38(MAPK) phosphorylation was altered under any experimental conditions. Taken together, the present study extends the available data on the mechanisms that underlie the antidepressant action of agmatine by showing an antidepressant-like effect following sub-chronic administration. In addition, our results are the first to demonstrate the ability of agmatine to elicit the activation of cellular signaling pathways associated with neuroplasticity/cell survival and the inhibition of signaling pathways associated with cell death in the hippocampus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

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Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

1994-11-04

Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

Science.gov (United States)

Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

2012-01-01

Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

LincRNA-p21 inhibits invasion and metastasis of hepatocellular carcinoma through miR-9/E-cadherin cascade signaling pathway molecular mechanism

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Ding G

2017-06-01

Full Text Available Gangqiang Ding, Zhen Peng, Jia Shang, Yi Kang, Huibin Ning, Chongshan Mao Department of Infectious Diseases, People’s Hospital of Zhengzhou University, Henan Provincial People’s Hospital, Zhengzhou, China Abstract: In the previous study, it was found that long intergenic noncoding RNA-p21 (lincRNA-p21 was downregulated in hepatocellular carcinoma (HCC and lincRNA-p21 overexpression inhibited tumor invasion through inducing epithelial–mesenchymal transition. However, the underlying mechanism was not fully elaborated. In this study, lincRNA-p21 expression was measured in 12 paired HCC and nontumor adjacent normal tissues by ­quantitative real-time polymerase chain reaction. The effects of lincRNA-p21 on HCC cells were studied using lentivirus expressing lincRNA-p21 vector in vitro. The association between lincRNA-p21 level and miR-9 level was tested with the Spearman rank correlation. The effects of miR-9 on HCC cells were studied by using miR-9 inhibitor in vitro. Luciferase assay was used to validate the target of miR-9. The results showed that lincRNA-p21 was downregulated in human HCC tissues and cell lines. LincRNA-p21 overexpression significantly inhibited HCC cell migration and invasion in vitro. Besides, lincRNA-p21 negatively regulated miR-9 expression level, and miR-9 was upregulated in human HCC tissues and cells. MiR-9 knockdown inhibited HCC cell migration and invasion in vitro. Finally, the luciferase assay results showed that E-cadherin was a direct target of miR-9. The expression level of E-cadherin was found to be regulated by lincRNA-p21 and miR-9. Altogether, the results suggested that lincRNA-p21 inhibits migration and invasion of HCC cells through regulating miR-9-mediated E-cadherin cascade signaling pathway. Keywords: hepatocellular carcinoma, lincRNA-p21, miR-9, E-cadherin, epithelial–mesenchymal transition

Molecular Analysis of a Multistep Lung Cancer Model Induced by Chronic Inflammation Reveals Epigenetic Regulation of p16, Activation of the DNA Damage Response Pathway

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David Blanco

2007-10-01

Full Text Available The molecular hallmarks of inflammation-mediated lung carcinogenesis have not been fully clarified, mainly due to the scarcity of appropriate animal models. We have used a silica-induced multistep lung carcinogenesis model driven by chronic inflammation to study the evolution of molecular markers, genetic alterations. We analyzed markers of DNA damage response (DDR, proliferative stress, telomeric stress: δ-H2AX, p16, p53, TERT. Lung cancer-related epigenetic, genetic alterations, including promoter hypermethylation status of p16(CDKN2A, APC, CDH13, Rassf1, Nore1A, as well as mutations of Tp53, epidermal growth factor receptor, K-ras, N-ras, c-H-ras, have been also studied. Our results showed DDR pathway activation in preneoplastic lesions, in association with inducible nitric oxide synthase, p53 induction. p16 was also induced in early tumorigenic progression, was inactivated in bronchiolar dysplasias, tumors. Remarkably, lack of mutations of Ras, epidermal growth factor receptor, a very low frequency of Tp53 mutations suggest that they are not required for tumorigenesis in this model. In contrast, epigenetic alterations in p16(CDKN2A, CDH13, APC, but not in Rassf1, Nore1A, were clearly observed. These data suggest the existence of a specific molecular signature of inflammation-driven lung carcinogenesis that shares some, but not all, of the molecular landmarks of chemically induced lung cancer.

Nur77 inhibits oxLDL induced apoptosis of macrophages via the p38 MAPK signaling pathway

International Nuclear Information System (INIS)

Shao, Qin; Han, Fei; Peng, Shi; He, Ben

2016-01-01

The interaction between macrophages and oxLDL plays a crucial role in the initiation and progression of atherosclerosis. As a key initiator in a number of plaque promoting processes, oxLDL induces variable effects such as cell apoptosis or proliferation. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it is of importance in vascular inflammation resulting in atherosclerosis, but whether Nur77 induction is detrimental or protective is unclear. In our study, we explore the role of Nur77 in the regulation of oxLDL-induced macrophage apoptosis and the signaling pathways that are involved. We found that oxLDL induced Nur77 expression in a dose and time dependent fashion, and cell viability was decreased in parallel. To determine whether Nur77 induction contributes to the loss of cell viability or is a protective mechanism, the effect of Nur77 overexpression was examined. Importantly, Nur77 overexpression inhibited the oxLDL-induced decrease of cell viability, inhibited the production of apoptotic bodies and restored DNA synthesis following oxLDL exposure. Furthermore, we found that Nur77 induction is mediated through the p38 MAPK signaling pathway. After pretreatment with SB203580, cell viability was decreased, the expression of CyclinA2 and PCNA was attenuated and the percentage of cell apoptosis was enhanced. Likewise, Nur77 overexpression increased the expression of the cell cycle genes PCNA and p21, and attenuated the increase in caspase-3. On the other hand, knockdown of Nur77 expression by specific siRNA resulted in the increased expression of caspase 3. The results demonstrate that Nur77 is induced by oxLDL via the p38 MAPK signaling pathway, which is involved in the regulation of cell survival. Nur77 enhanced cell survival via suppressing apoptosis, without affecting cell proliferation of activated macrophages, which may be beneficial in patients with atherosclerosis. - Highlights: • oxLDL could induce Nur77

Nur77 inhibits oxLDL induced apoptosis of macrophages via the p38 MAPK signaling pathway

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Shao, Qin; Han, Fei; Peng, Shi; He, Ben, E-mail: heben@medmail.com.cn

2016-03-18

The interaction between macrophages and oxLDL plays a crucial role in the initiation and progression of atherosclerosis. As a key initiator in a number of plaque promoting processes, oxLDL induces variable effects such as cell apoptosis or proliferation. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it is of importance in vascular inflammation resulting in atherosclerosis, but whether Nur77 induction is detrimental or protective is unclear. In our study, we explore the role of Nur77 in the regulation of oxLDL-induced macrophage apoptosis and the signaling pathways that are involved. We found that oxLDL induced Nur77 expression in a dose and time dependent fashion, and cell viability was decreased in parallel. To determine whether Nur77 induction contributes to the loss of cell viability or is a protective mechanism, the effect of Nur77 overexpression was examined. Importantly, Nur77 overexpression inhibited the oxLDL-induced decrease of cell viability, inhibited the production of apoptotic bodies and restored DNA synthesis following oxLDL exposure. Furthermore, we found that Nur77 induction is mediated through the p38 MAPK signaling pathway. After pretreatment with SB203580, cell viability was decreased, the expression of CyclinA2 and PCNA was attenuated and the percentage of cell apoptosis was enhanced. Likewise, Nur77 overexpression increased the expression of the cell cycle genes PCNA and p21, and attenuated the increase in caspase-3. On the other hand, knockdown of Nur77 expression by specific siRNA resulted in the increased expression of caspase 3. The results demonstrate that Nur77 is induced by oxLDL via the p38 MAPK signaling pathway, which is involved in the regulation of cell survival. Nur77 enhanced cell survival via suppressing apoptosis, without affecting cell proliferation of activated macrophages, which may be beneficial in patients with atherosclerosis. - Highlights: • oxLDL could induce Nur77

Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway

International Nuclear Information System (INIS)

Tsubaki, Masanobu; Satou, Takao; Itoh, Tatsuki; Imano, Motohiro; Ogaki, Mitsuhiko; Yanae, Masashi; Nishida, Shozo

2012-01-01

Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion, and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma. -- Highlights: ► We investigated whether YM529/ONO-5920 inhibited tumor metastasis in osteosarcoma. ► YM529/ONO-5920 inhibited metastasis, cell migration, invasion, and adhesion. ► YM529/ONO-5920 suppressed Ras signalings. ► YM529/ONO-5920

Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

International Nuclear Information System (INIS)

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Audemard, Eric; Montermini, Laura; Meehan, Brian; Rak, Janusz

2014-01-01

Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells

Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

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Lee, Tae Hoon; Chennakrishnaiah, Shilpa [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Audemard, Eric [McGill University and Genome Quebec Innovation Centre, Montreal, Quebec (Canada); Montermini, Laura; Meehan, Brian [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Rak, Janusz, E-mail: janusz.rak@mcgill.ca [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

2014-08-22

Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.

Upregulation of c-mesenchymal epithelial transition expression and RAS mutations are associated with late lung metastasis and poor prognosis in colorectal carcinoma.

Science.gov (United States)

Liu, Jianhua; Zeng, Weiqiang; Huang, Chengzhi; Wang, Junjiang; Xu, Lishu; Ma, Dong

2018-05-01

The present study aimed to investigate whether c-mesenchymal epithelial transition factor (C-MET) overexpression combined with RAS (including KRAS, NRAS and HRAS ) or BRAF mutations were associated with late distant metastases and the prognosis of patients with colorectal cancer (CRC). A total of 374 patients with stage III CRC were classified into 4 groups based on RAS/BRAF and C-MET status for comprehensive analysis. Mutations in RAS / BRAF were determined using Sanger sequencing and C-MET expression was examined using immunohistochemistry. The associations between RAS/BRAF mutations in combination with C-MET overexpression and clinicopathological variables including survival were evaluated. In addition, their predictive value for late distant metastases were statistically analyzed via logistic regression and receiver operating characteristic analysis. Among 374 patients, mutations in KRAS, NRAS, HRAS, BRAF and C-MET overexpression were observed in 43.9, 2.4, 0.3, 5.9 and 71.9% of cases, respectively. Considering RAS/BRAF mutations and C-MET overexpression, vascular invasion (P=0.001), high carcino-embryonic antigen level (P=0.031) and late distant metastases (PC-MET overexpression, although they were both predictive factors for adverse prognosis. Further logistic regression suggested that RAS/BRAF mutations and C-MET overexpression may predict late distant metastases. In conclusion, RAS/BRAF mutations and C-MET overexpression may serve as predictive indicators for metastatic behavior and poor prognosis of CRC.

Genetic and Molecular Analysis of Suppressors of Ras Mutations

National Research Council Canada - National Science Library

Sieburth, Derek

1998-01-01

.... elegans vulval development. We describe the identification and characterization of a novel gene, sur-8, that functions to regulate a receptor tyrosine kinase-Ras-MAP kinase-mediated signal transduction pathway during C...

Involvement of H- and N-Ras isoforms in transforming growth factor-β1-induced proliferation and in collagen and fibronectin synthesis

International Nuclear Information System (INIS)

Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Garcia-Cenador, Begona; Santos, Eugenio; Lopez-Novoa, Jose M.

2006-01-01

Transforming growth factor β1 (TGF-β1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-β and Ras signaling pathways are closely related: TGF-β1 overcomes Ras mitogenic effects and Ras counteracts TGF-β signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-β1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras -/- /N-ras -/- ) isoforms and from heterozygote mice (H-ras +/- /N-ras +/- ). ECM synthesis is increased in basal conditions in H-ras -/- /N-ras -/- fibroblasts, this increase being higher after stimulation with TGF-β1. TGF-β1-induced fibroblast proliferation is smaller in H-ras -/- /N-ras -/- than in H-ras +/- /N-ras +/- fibroblasts. Erk activation is decreased in H-ras -/- /N-ras -/- fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras

Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

Science.gov (United States)

Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

2016-06-01

Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations

International Nuclear Information System (INIS)

Preto, Ana; Soares, Paula; Sobrinho-Simões, Manuel; Gonçalves, Joana; Rebocho, Ana P; Figueiredo, Joana; Meireles, Ana M; Rocha, Ana S; Vasconcelos, Helena M; Seca, Hugo; Seruca, Raquel

2009-01-01

Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds. Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot. Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF V600E mutation. In BRAF V600E mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27 Kip1 . Specific inhibition of BRAF by RNAi in cells with BRAF V600E mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF V600E mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2. Our results in thyroid cancer cells, namely those harbouring BRAF V600E mutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF V600E mutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and

Ras oncogenes in oral cancer: the past 20 years.

Science.gov (United States)

Murugan, Avaniyapuram Kannan; Munirajan, Arasambattu Kannan; Tsuchida, Nobuo

2012-05-01

Oral squamous cell carcinoma (OSCC) of head and neck is associated with high morbidity and mortality in both Western and Asian countries. Several risk factors for the development of oral cancer are very well established, including tobacco chewing, betel quid, smoking, alcohol drinking and human papilloma virus (HPV) infection. Apart from these risk factors, many genetic factors such as oncogenes, tumor suppressor genes and regulatory genes are identified to involve in oral carcinogenesis with these risk factors dependent and independent manner. Ras is one of the most frequently genetically deregulated oncogene in oral cancer. In this review, we analyze the past 22years of literature on genetic alterations such as mutations and amplifications of the isoforms of the ras oncogene in oral cancer. Further, we addressed the isoform-specific role of the ras in oral carcinogenesis. We also discussed how targeting the Akt and MEK, downstream effectors of the PI3K/Akt and MAPK pathways, respectively, would probably pave the possible molecular therapeutic target for the ras driven tumorigenesis in oral cancer. Analysis of these ras isoforms may critically enlighten specific role of a particular ras isoform in oral carcinogenesis, enhance prognosis and pave the way for isoform-specific molecular targeted therapy in OSCC. Copyright © 2011 Elsevier Ltd. All rights reserved.

RasGRP3 regulates the migration of glioma cells via interaction with Arp3

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Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M.; Blumberg, Peter M.; Brodie, Chaya

2015-01-01

Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. PMID:25682201

Genetic and Molecular Analysis of Suppressors of Ras Mutations

National Research Council Canada - National Science Library

Sieburth, Derek

1999-01-01

.... elegans vulvaZ development. We describe the identification and characterization of a novel gene, sur-8, that functions to regulate a receptor tyrosine kinase-Ras-MAp kinase- mediated signal transduction pathway during C...

CBX7 regulates stem cell-like properties of gastric cancer cells via p16 and AKT-NF-κB-miR-21 pathways.

Science.gov (United States)

Ni, Su-Jie; Zhao, Li-Qin; Wang, Xiao-Feng; Wu, Zhen-Hua; Hua, Rui-Xi; Wan, Chun-Hua; Zhang, Jie-Yun; Zhang, Xiao-Wei; Huang, Ming-Zhu; Gan, Lu; Sun, Hua-Lin; Dimri, Goberdhan P; Guo, Wei-Jian

2018-02-08

Chromobox protein homolog 7 (CBX7), a member of the polycomb group (PcG) family of proteins, is involved in the regulation of cell proliferation and cancer progression. PcG family members, such as BMI, Mel-18, and EZH2, are integral constituents of the polycomb repressive complexes (PRCs) and have been known to regulate cancer stem cell (CSC) phenotype. However, the role of other PRCs' constituents such as CBX7 in the regulation of CSC phenotype remains largely elusive. This study was to investigate the role of CBX7 in regulating stem cell-like properties of gastric cancer and the underlying mechanisms. Firstly, the role of CBX7 in regulating stem cell-like properties of gastric cancer was investigated using sphere formation, Western blot, and xenograft tumor assays. Next, RNA interference and ectopic CBX7 expression were employed to determine the impact of CBX7 on the expression of CSC marker proteins and CSC characteristics. The expression of CBX7, its downstream targets, and stem cell markers were analyzed in gastric stem cell spheres, common cancer cells, and gastric cancer tissues. Finally, the pathways by which CBX7 regulates stem cell-like properties of gastric cancer were explored. We found that CBX7, a constituent of the polycomb repressive complex 1 (PRC1), plays an important role in maintaining stem cell-like characteristics of gastric cancer cells via the activation of AKT pathway and the downregulation of p16. Spearman rank correlation analysis showed positive correlations among the expression of CBX7 and phospho-AKT (pAKT), stem cell markers OCT-4, and CD133 in gastric cancer tissues. In addition, CBX7 was found to upregulate microRNA-21 (miR-21) via the activation of AKT-NF-κB pathway, and miR-21 contributes to CBX7-mediated CSC characteristics. CBX7 positively regulates stem cell-like characteristics of gastric cancer cells by inhibiting p16 and activating AKT-NF-κB-miR-21 pathway.

Trabecular meshwork ECM remodeling in glaucoma: could RAS be a target?

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Agarwal, Puneet; Agarwal, Renu

2018-06-14

Disturbances of extracellular matrix (ECM) homeostasis in trabecular meshwork (TM) cause increased aqueous outflow resistance leading to elevated intraocular pressure (IOP) in glaucomatous eyes. Therefore, restoration of ECM homeostasis is a rational approach to prevent disease progression. Since renin-angiotensin system (RAS) inhibition positively alters ECM homeostasis in cardiovascular pathologies involving pressure and volume overload, it is likely that RAS inhibitors reduce IOP primarily by restoring ECM homeostasis. Areas covered: Current evidence showing the presence of RAS components in ocular tissue and its role in regulating aqueous humor dynamics is briefly summarized. The role of RAS in ECM remodeling is discussed both in terms of its effects on ECM synthesis and its breakdown. The mechanisms of ECM remodeling involving interactions of RAS with transforming growth factor-β, Wnt/β-catenin signaling, bone morphogenic proteins, connective tissue growth factor, and matrix metalloproteinases in ocular tissue are discussed. Expert opinion: Current literature strongly indicates a significant role of RAS in ECM remodeling in TM of hypertensive eyes. Hence, IOP-lowering effect of RAS inhibitors may primarily be attributed to restoration of ECM homeostasis in aqueous outflow pathways rather than its vascular effects. However, the mechanistic targets for RAS inhibitors have much wider distribution and consequences, which remain relatively unexplored in TM.

A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK

International Nuclear Information System (INIS)

Abe, Yasuhito; Takeuchi, Takashi; Imai, Yoshinori; Murase, Ryuichi; Kamei, Yoshiaki; Fujibuchi, Taketsugu; Matsumoto, Suguru; Ueda, Norifumi; Ogasawara, Masahito; Shigemoto, Kazuhiro; Kito, Katsumi

2006-01-01

PRPK phosphorylates serine-15 residue of p53 and enhances transcriptional activity. PRPK possesses a bipartite nuclear localization signal and localizes in nucleus when over-expressed in cells. However, intrinsic PRPK localizes mainly in the cytosol in situ. While studying the mechanisms in the distribution of intrinsic PRPK, we identified a PRPK binding protein, an ubiquitously expressed Small Ras-like GTPase, Rab1c, also named Ray or Rab35. The over-expressed Ray was distributed in the nucleus, cytosol, and cell membrane. Both Ray wild type and GTP-restrictively binding mutant Ray-Q67L, but not guanine nucleotide unstable binding mutant Ray-N120I, partially distributed the over-expressed PRPK to the cytosol and also suppressed the PRPK-induced p53-transcriptional activity profoundly. A Small Ras-like GTPase protein Ray was thus indicated to modulate p53 transcriptional activity of PRPK

Evodiamine synergizes with doxorubicin in the treatment of chemoresistant human breast cancer without inhibiting P-glycoprotein.

Directory of Open Access Journals (Sweden)

Shengpeng Wang

Full Text Available Drug resistance is one of the main hurdles for the successful treatment of breast cancer. The synchronous targeting of apoptosis resistance and survival signal transduction pathways may be a promising approach to overcome drug resistance. In this study, we determined that evodiamine (EVO, a major constituent of the Chinese herbal medicine Evodiae Fructus, could induce apoptosis of doxorubicin (DOX-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells in a caspase-dependent manner, as confirmed by significant increases of cleaved poly(ADP-ribose polymerase (PARP, caspase-7/9, and caspase activities. Notably, the reversed phenomenon of apoptosis resistance by EVO might be attributed to its ability to inhibit the Ras/MEK/ERK pathway and the expression of inhibitors of apoptosis (IAPs. Furthermore, our results indicated that EVO enhanced the apoptotic action of DOX by inhibiting the Ras/MEK/ERK cascade and the expression of IAPs without inhibiting the expression and activity of P-glycoprotein (P-gp. Taken together, our data indicate that EVO, a natural product, may be useful applied alone or in combination with DOX for the treatment of resistant breast cancer.

Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway

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D' Ambrosio, Steven M. [Department of Radiology, College of Medicine, The Ohio State University, Columbus, OH 43210 (United States); Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210 (United States); Han, Chunhua [Department of Radiology, College of Medicine, The Ohio State University, Columbus, OH 43210 (United States); Pan, Li; Douglas Kinghorn, A. [Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210 (United States); Ding, Haiming, E-mail: ding.29@osu.edu [Department of Radiology, College of Medicine, The Ohio State University, Columbus, OH 43210 (United States)

2011-06-10

Highlights: {yields} The aliphatic acetogenins [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] (1) and [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate] (2) isolated from avocado fruit inhibit phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). {yields} Aliphatic acetogenin 2, but not 1, prevents EGF-induced activation of EGFR (Tyr1173). {yields} Combination of both aliphatic acetogenins synergistically inhibits c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation and human oral cancer cell proliferation. {yields} The potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins targeting two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. {yields} Providing a double hit on a critical cancer pathway such as EGFR/RAS/RAF/MEK/ERK1/2 by phytochemicals like those found in avocado fruit could lead to more effective approach toward cancer prevention. -- Abstract: Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003) was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compounds 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not

Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway

International Nuclear Information System (INIS)

D'Ambrosio, Steven M.; Han, Chunhua; Pan, Li; Douglas Kinghorn, A.; Ding, Haiming

2011-01-01

Highlights: → The aliphatic acetogenins [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] (1) and [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate] (2) isolated from avocado fruit inhibit phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). → Aliphatic acetogenin 2, but not 1, prevents EGF-induced activation of EGFR (Tyr1173). → Combination of both aliphatic acetogenins synergistically inhibits c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation and human oral cancer cell proliferation. → The potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins targeting two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. → Providing a double hit on a critical cancer pathway such as EGFR/RAS/RAF/MEK/ERK1/2 by phytochemicals like those found in avocado fruit could lead to more effective approach toward cancer prevention. -- Abstract: Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003) was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compounds 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not compound 1, prevented EGF

ATXN1L, CIC, and ETS Transcription Factors Modulate Sensitivity to MAPK Pathway Inhibition | Office of Cancer Genomics

Science.gov (United States)

Intrinsic resistance and RTK-RAS-MAPK pathway reactivation has limited the effectiveness of MEK and RAF inhibitors (MAPKi) in RAS- and RAF-mutant cancers. To identify genes that modulate sensitivity to MAPKi, we performed genome-scale CRISPR-Cas9 loss-of-function screens in two KRAS mutant pancreatic cancer cell lines treated with the MEK1/2 inhibitor trametinib. Loss of CIC, a transcriptional repressor of ETV1, ETV4, and ETV5, promoted survival in the setting of MAPKi in cancer cells derived from several lineages.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

Science.gov (United States)

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

Directory of Open Access Journals (Sweden)

Edwards Jeremy

2003-07-01

Full Text Available Abstract Background The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH, in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2. Results Using polony technology, Panc-1 was determined to possess only one copy of p53, which possessed a mutation in codon 273, and two copies of K-ras2, one wildtype and one with a mutation in codon 12. To further demonstrate the general approach of this method, polonies were also used to detect K-ras2 mutations in the pancreatic cell lines, AsPc-1 and CAPAN-1. Conclusions In conclusion, we have developed an assay that can detect mutations in hotspots of p53 and K-ras2 as well as diagnose LOH in these same genes.

[Expressions of Ras and Sos1 in epithelial ovarian cancer tissues and their clinical significance].

Science.gov (United States)

Xiao, Zheng-Hua; Linghu, Hua; Liu, Qian-Fen

2016-11-20

To detect the expressions of Ras and Sos1 proteins in human epithelial ovarian cancer (EOC) tissues and explore their correlation with the clinicopathological features of the patients. The expressions of Ras and Sos1 proteins were detected immunohistochemically in 62 EOC tissues, 5 borderline ovarian cancer tissues, 15 benign epithelial ovarian neoplasm tissues, and 18 normal ovarian tissues. The EOC tissues showed significantly higher expression levels of both Ras and Sos1 than the other tissues tested (Ptissues, Ras and Sos1 proteins were expressed mostly on the cell membrane and in the cytoplasm. The expression level of Ras was correlated with pathological types of the tumor (Ptissue-specific variation of Ras expression can lend support to a specific diagnosis of ovarian serous adenocarcinoma. The association of Ras and Sos1 protein expression with the tumor-free survival time of the patients awaits further investigation with a larger sample size.

Common genetic polymorphisms of microRNA biogenesis pathway genes and breast cancer survival

International Nuclear Information System (INIS)

Sung, Hyuna; Ahn, Sei-Hyun; Kang, Daehee; Jeon, Sujee; Lee, Kyoung-Mu; Han, Sohee; Song, Minkyo; Choi, Ji-Yeob; Park, Sue K; Yoo, Keun-Young; Noh, Dong-Young

2012-01-01

Although the role of microRNA’s (miRNA’s) biogenesis pathway genes in cancer development and progression has been well established, the association between genetic variants of this pathway genes and breast cancer survival is still unknown. We used genotype data available from a previously conducted case–control study to investigate association between common genetic variations in miRNA biogenesis pathway genes and breast cancer survival. We investigated the possible associations between 41 germ-line single-nucleotide polymorphisms (SNPs) and both disease free survival (DFS) and overall survival (OS) among 488 breast cancer patients. During the median follow-up of 6.24 years, 90 cases developed disease progression and 48 cases died. Seven SNPs were significantly associated with breast cancer survival. Two SNPs in AGO2 (rs11786030 and rs2292779) and DICER1 rs1057035 were associated with both DFS and OS. Two SNPs in HIWI (rs4759659 and rs11060845) and DGCR8 rs9606250 were associated with DFS, while DROSHA rs874332 and GEMIN4 rs4968104 were associated with only OS. The most significant association was observed in variant allele of AGO2 rs11786030 with 2.62-fold increased risk of disease progression (95% confidence interval (CI), 1.41-4.88) and in minor allele homozygote of AGO2 rs2292779 with 2.94-fold increased risk of death (95% CI, 1.52-5.69). We also found cumulative effects of SNPs on DFS and OS. Compared to the subjects carrying 0 to 2 high-risk genotypes, those carrying 3 or 4–6 high-risk genotypes had an increased risk of disease progression with a hazard ratio of 2.16 (95% CI, 1.18- 3.93) and 4.47 (95% CI, 2.45- 8.14), respectively (P for trend, 6.11E-07). Our results suggest that genetic variants in miRNA biogenesis pathway genes may be associated with breast cancer survival. Further studies in larger sample size and functional characterizations are warranted to validate these results

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

Energy Technology Data Exchange (ETDEWEB)

Su, L.-N.; Little, J.B. (Harvard School of Public Health, Boston, MA (United States))

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author).

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

International Nuclear Information System (INIS)

Su, L.-N.; Little, J.B.

1992-01-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author)

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Energy Technology Data Exchange (ETDEWEB)

Copp& #233; , Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith

2008-10-24

Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

Mutated N-ras does not induce p19 arf in CO25 cell line | Saleh ...

African Journals Online (AJOL)

The mouse cell line (CO25) used in this study was transfected with a glucocorticoid inducible mutated human N-ras oncogene under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumors virus long terminal repeat MMTV-LTR. This study was aimed to investigate the expression of p19arf and ...

The RAS Initiative

Science.gov (United States)

NCI established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to ultimately create effective, new therapies for RAS-related cancers.

PTEN expression is consistent in colorectal cancer primaries and metastases and associates with patient survival

International Nuclear Information System (INIS)

Atreya, Chloe E; Sangale, Zaina; Xu, Nafei; Matli, Mary R; Tikishvili, Eliso; Welbourn, William; Stone, Steven; Shokat, Kevan M; Warren, Robert S

2013-01-01

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates the phosphoinositide-3-kinase (PI3K) signaling pathway. In colorectal cancer (CRC), observed frequencies of loss of PTEN expression, concordant expression in primary tumors and metastases, and the association of PTEN status with outcome vary markedly by detection method. We determined the degree to which PTEN expression is consistent in 70 matched human CRC primaries and liver metastases using a validated immunohistochemistry assay. We found loss of PTEN expression in 12.3% of assessable CRC primaries and 10.3% of assessable liver metastases. PTEN expression (positive or negative) was concordant in 98% of matched colorectal primaries and liver metastases. Next we related PTEN status to mutations in RAS and PI3K pathway genes (KRAS, NRAS, BRAF, and PIK3CA) and to overall survival (OS). PTEN expression was not significantly associated with the presence or absence of mutations in RAS or PI3K pathway genes. The median OS of patients whose tumors did not express PTEN was 9 months, compared to 49 months for patients whose tumors did express PTEN (HR = 6.25, 95% confidence intervals (CI) (1.98, 15.42), P = 0.0017). The association of absent PTEN expression with increased risk of death remained significant in multivariate analysis (HR = 6.31, 95% CI (2.03, 17.93), P = 0.0023). In summary, PTEN expression was consistent in matched CRC primaries and in liver metastases. Therefore, future investigations of PTEN in metastatic CRC can use primary tumor tissue. In patients with liver-only metastases, loss of PTEN expression predicted poor OS. We observed concordant PTEN expression in 98% of colorectal cancer (CRC) primary and liver metastasis pairs using a validated immunohistochemistry assay. Consistent PTEN expression at both disease sites is significant because tumor tissue is usually available from CRC primaries but not metastases. Loss of PTEN expression associated with poor survival of

RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

Science.gov (United States)

Fang, Bingliang

2016-01-01

Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology

A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.

Directory of Open Access Journals (Sweden)

Dariel Ashton-Beaucage

2014-03-01

Full Text Available The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing

Identification and preliminary characterization of protein-cysteine farnesyltransferase

International Nuclear Information System (INIS)

Manne, V.; Roberts, D.; Tobin, A.; O'Rourke, E.; Barbacid, M.; De Virgilio, M.; Meyers, C.; Ahmed, N.; Kurz, B.; Resh, M.; Kung, Hsiang-Fu

1990-01-01

Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2 Valendash19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an onocogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, the authors describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway

Regulation of the PI3K pathway through a p85a monomer-homodimer equilibrium

KAUST Repository

Aljedani, Safia Salim Eid

2017-01-08

The phosphatidylinositol-3-kinase a (PI3Ka) is heterodimeric enzyme that is composed of p85a regulatory subunit and a p110a catalytic subunit. PI3Ka plays a key role in cell survival, growth and differentation. Owing to its role as a key regulator, the PI3Ka pathway is the most frequently mutated pathway in human cancers, and is targeted by many viruses to insure their survival and successful reproduction. Previous studies have shown that the equilibrium of p85 monomers and dimers regulates the PI3K pathway, suggesting that interrupting this equilibrium could lead to disease development. Moreover, studies suggest that the p85a monomers and dimers have opposing effects on PI3Ka signaling as only the p85a dimers bind to the PTEN phosphatase, whereas p85a monomers bind to the catalytic p110 subunit. However, the mechanism for dimerisation is controversial, and it is unknown why PTEN or p110a bind only dimer or monomer. Therefore, we combine molecular biology, biophsical, computational and structural methods to investigate the suprosingly complex p85 dimerisation mechanism and its control by ligands. Results may inspire novel theraputic approaches.

Novel adapter proteins that link the human GM-CSF receptor to the phosphatidylino-sitol 3-kinase and Shc/Grb2/ras signaling pathways.

Science.gov (United States)

Jücker, M; Feldman, R A

1996-01-01

We have used a human GM-CSF-dependent hematopoietic cell line that responds to physiological concentrations of hGM-CSF to analyze a set of signaling events that occur in normal myelopoiesis and whose deregulation may lead to leukemogenesis. Stimulation of these cells with hGM-CSF induced the assembly of multimeric complexes that contained known and novel phosphotyrosyl proteins. One of the new proteins was a major phosphotyrosyl substrate of 76-85 kDa (p80) that was directly associated with the p85 subunit of phosphatidylinositol (PI) 3-kinase through the SH2 domains of p85. p80 also associated with the beta subunit of the activated hGM-CSF receptor, and assembly of this complex correlated with activation of PI 3-kinase. A second phosphotyrosyl protein we identified, p140, associated with the Shc and Grb2 adapter proteins by direct binding to a novel phosphotyrosine-interacting domain located at the N-terminus of Shc. and to the SH3 domains of Grb2, respectively. The Shc/p140/Grb2 complex was found to be constitutively activated in acute myeloid leukemia cells, indicating that activation of this pathway may be a necessary step in the development of some leukemias. The p80/p85/PI 3-kinase and the Shc/Grb2/p140 complexes were tightly associated with Src family kinases, which were prime candidates for phosphorylation of Shc, p80, p140 and other phosphotyrosyl substrates present in these complexes. Our studies suggest that p80 and p140 may link the hGM-CSF receptor to the PI 3-kinase and Shc/Grb2/ras signaling pathways, respectively, and that abnormal activation of hGM-CSF-dependent targets may play a role in leukemogenesis.

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

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Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

Science.gov (United States)

Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2018-05-01

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

Increased p53 and decreased p21 accompany apoptosis induced by ultraviolet radiation in the nervous system of a crustacean

International Nuclear Information System (INIS)

Hollmann, Gabriela; Linden, Rafael; Giangrande, Angela; Allodi, Silvana

2016-01-01

Highlights: • The paper characterizes molecular pathways of cell responses to environmental doses of UV in brain tissue of a crab species. • The UV radiation changes levels of proteins which trigger apoptotic or cell cycle arrest pathways and also it changes neurotrophins which lead to apoptosis of neural cell in the central nervous system (CNS) of the crab Ucides cordatus. • The UVB wavelengths in the solar simulator damaged the DNA, either directly or indirectly, by increasing ROS, and induced the increase of p53 and AKT, which blocked p21 and increased the expression of activated caspase-3, triggering apoptosis. The signs of death increased the expression of neurotrophins (BDNF and GDNF), which continued to stimulate the apoptosis signaling mediated by caspase-3. • In the brain of the crab U. cordatus, p53/p21 relationship in response to UV radiation is different from that of most mammals. - Abstract: Ultraviolet (UV) radiation can produce biological damage, leading the cell to apoptosis by the p53 pathway. This study evaluated some molecular markers of the apoptosis pathway induced by UVA, UVB and UVA+ UVB (Solar Simulator, SIM) in environmental doses, during five consecutive days of exposure, in the brain of the crab Ucides cordatus. We evaluated the central nervous system (CNS) by immunoblotting the content of proteins p53, p21, phosphorylated AKT, BDNF, GDNF, activated caspase-3 (C3) and phosphohistone H3 (PH3); and by immunohistochemical tests of the cells labeled for PH3 and C3. After the fifth day of exposure, UVB radiation and SIM increased the protein content of p53, increasing the content of AKT and, somehow, blocking p21, increasing the content of activated caspase-3, which led the cells to apoptosis. The signs of death affected the increase in neurotrophins, such as BDNF and GDNF, stimulating the apoptotic cascade of events. Immunohistochemical assays and immunoblotting showed that apoptosis was present in the brains of all UV groups, while

Increased p53 and decreased p21 accompany apoptosis induced by ultraviolet radiation in the nervous system of a crustacean

Energy Technology Data Exchange (ETDEWEB)

Hollmann, Gabriela, E-mail: gabrielahollmann@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil); Linden, Rafael, E-mail: rlinden@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil); Giangrande, Angela, E-mail: angela.giangrande@igbmc.fr [Institut de Génétique et de Biologie Moléculaire et Cellulaire-IGBMC, INSERM, Strasbourg (France); Allodi, Silvana, E-mail: sallodi@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil)

2016-04-15

Highlights: • The paper characterizes molecular pathways of cell responses to environmental doses of UV in brain tissue of a crab species. • The UV radiation changes levels of proteins which trigger apoptotic or cell cycle arrest pathways and also it changes neurotrophins which lead to apoptosis of neural cell in the central nervous system (CNS) of the crab Ucides cordatus. • The UVB wavelengths in the solar simulator damaged the DNA, either directly or indirectly, by increasing ROS, and induced the increase of p53 and AKT, which blocked p21 and increased the expression of activated caspase-3, triggering apoptosis. The signs of death increased the expression of neurotrophins (BDNF and GDNF), which continued to stimulate the apoptosis signaling mediated by caspase-3. • In the brain of the crab U. cordatus, p53/p21 relationship in response to UV radiation is different from that of most mammals. - Abstract: Ultraviolet (UV) radiation can produce biological damage, leading the cell to apoptosis by the p53 pathway. This study evaluated some molecular markers of the apoptosis pathway induced by UVA, UVB and UVA+ UVB (Solar Simulator, SIM) in environmental doses, during five consecutive days of exposure, in the brain of the crab Ucides cordatus. We evaluated the central nervous system (CNS) by immunoblotting the content of proteins p53, p21, phosphorylated AKT, BDNF, GDNF, activated caspase-3 (C3) and phosphohistone H3 (PH3); and by immunohistochemical tests of the cells labeled for PH3 and C3. After the fifth day of exposure, UVB radiation and SIM increased the protein content of p53, increasing the content of AKT and, somehow, blocking p21, increasing the content of activated caspase-3, which led the cells to apoptosis. The signs of death affected the increase in neurotrophins, such as BDNF and GDNF, stimulating the apoptotic cascade of events. Immunohistochemical assays and immunoblotting showed that apoptosis was present in the brains of all UV groups, while

Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis.

Science.gov (United States)

Overmeyer, Jean H; Kaul, Aparna; Johnson, Erin E; Maltese, William A

2008-06-01

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.

Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFβ cell cycle arrest

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Harrison David J

2007-11-01

Full Text Available Abstract Background TGFβ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFβ in disease. We asked how Rb-deficiency would affect responses to TGFβ-induced cell cycle arrest. Results Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16INK4A and P21Cip and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFβ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21Cip1 and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21Cip1. Hepatocytes deficient in p53 or p21Cip1 showed diminished growth inhibition by TGFβ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21Cip and P53 work through the same pathway to regulate G1/S in response to TGFβ. In Rb-deficient cells however, p53 but not p21Cip deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity. Conclusion The present results show that otherwise genetically normal hepatocytes with disabled p53, p21Cip1 or Rb genes respond less well to the antiproliferative effects of TGFβ. As the function of

Predictive value of K-ras and PIK3CA in non-small cell lung cancer patients treated with EGFR-TKIs: a systemic review and meta-analysis

International Nuclear Information System (INIS)

Chen, Jie-Ying; Cheng, Ya-Nan; Han, Lei; Wei, Feng; Yu, Wen-Wen; Zhang, Xin-Wei; Cao, Shui; Yu, Jin-Pu

2015-01-01

A meta-analysis was performed to augment the insufficient data on the impact of mutative EGFR downstream phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on the clinical efficiency of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment of non-small cell lung cancer (NSCLC) patients. Network databases were explored in April, 2015. Papers that investigated the clinical outcomes of NSCLC patients treated with EGFR-TKIs according to the status of K-ras and/or PIK3CA gene mutation were included. A quantitative meta-analysis was conducted using standard statistical methods. Odds ratios (ORs) for objective response rate (ORR) and hazard ratios (HRs) for progression-free survival (PFS) and overall survival (OS) were calculated. Mutation in K-ras significantly predicted poor ORR [OR =0.22; 95% confidence interval (CI), 0.13-0.35], shorter PFS (HR =1.56; 95% CI, 1.27-1.92), and shorter OS (HR =1.59; 95% CI, 1.33-1.91) in NSCLC patients treated with EGFR-TKIs. Mutant PIK3CA significantly predicted shorter OS (HR =1.83; 95% CI, 1.05-3.20), showed poor ORR (OR =0.70; 95% CI, 0.22-2.18), and shorter PFS (HR =1.79; 95% CI, 0.91-3.53) in NSCLC patients treated with EGFR-TKIs. K-ras mutation adversely affected the clinical response and survival of NSCLC patients treated with EGFR-TKIs. PIK3CA mutation showed similar trends. In addition to EGFR, adding K-ras and PIK3CA as routine gene biomarkers in clinical genetic analysis is valuable to optimize the effectiveness of EGFR-TKI regimens and identify optimal patients who will benefit from EGFR-TKI treatment

Sequence analysis of the Ras-MAPK pathway genes SOS1, EGFR & GRB2 in silver foxes (Vulpes vulpes): candidate genes for hereditary hyperplastic gingivitis.

Science.gov (United States)

Clark, Jo-Anna B J; Tully, Sara J; Dawn Marshall, H

2014-12-01

Hereditary hyperplastic gingivitis (HHG) is an autosomal recessive disease that presents with progressive gingival proliferation in farmed silver foxes. Hereditary gingival fibromatosis (HGF) is an analogous condition in humans that is genetically heterogeneous with several known autosomal dominant loci. For one locus the causative mutation is in the Son of sevenless homologue 1 (SOS1) gene. For the remaining loci, the molecular mechanisms are unknown but Ras pathway involvement is suspected. Here we compare sequences for the SOS1 gene, and two adjacent genes in the Ras pathway, growth receptor bound protein 2 (GRB2) and epidermal growth factor receptor (EGFR), between HHG-affected and unaffected foxes. We conclude that the known HGF causative mutation does not cause HHG in foxes, nor do the coding regions or intron-exon boundaries of these three genes contain any candidate mutations for fox gum disease. Patterns of molecular evolution among foxes and other mammals reflect high conservation and strong functional constraints for SOS1 and GRB2 but reveal a lineage-specific pattern of variability in EGFR consistent with mutational rate differences, relaxed functional constraints, and possibly positive selection.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

Science.gov (United States)

Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

The prognostic value of simultaneous tumor and serum RAS/RAF mutations in localized colon cancer

DEFF Research Database (Denmark)

Brenner Thomsen, Caroline Emilie; Appelt, Ane Lindegaard; Andersen, Rikke Fredslund

2017-01-01

The impact of RAS/RAF mutations in localized colon cancer needs clarification. Based on analysis of tumor-specific DNA, this study aimed at elucidating the prognostic influence of mutational status in tumor and serum using an extended panel of mutations. The study retrospectively included 294.......0057), and disease-free survival (DFS) (HR = 2.18, 95%CI = 1.26-3.77, P = 0.0053). BRAF mutation in the serum and proficient mismatch repair (pMMR) protein in tumor also indicated significantly worse prognosis, OS (HR = 3.45, 95% CI = 1.52-7.85, P = 0.0032) and DFS (HR = 3.61, 95% CI = 1.70-7.67, P = 0...

Human Mut T Homolog 1 (MTH1): a roadblock for the tumor-suppressive effects of oncogenic RAS-induced ROS.

Science.gov (United States)

Rai, Priyamvada

2012-01-01

Oncogenic RAS-induced reactive oxygen species (ROS) trigger barriers to cell transformation and cancer progression through tumor-suppressive responses such as cellular senescence or cell death. We have recently shown that oncogenic RAS-induced DNA damage and attendant premature senescence can be prevented by overexpressing human MutT Homolog 1 (MTH1), the major mammalian detoxifier of the oxidized DNA precursor, 8-oxo-dGTP. Paradoxically, RAS-induced ROS are also able to participate in tumor progression via transformative processes such as mitogenic signaling, the epithelial-mesenchymal transition (EMT), anoikis inhibition, and PI3K/Akt-mediated survival signaling. Here we provide a preliminary insight into the influence of MTH1 levels on the EMT phenotype and Akt activation in RAS-transformed HMLE breast epithelial cells. Within this context, we will discuss the implications of MTH1 upregulation in oncogenic RAS-sustaining cells as a beneficial adaptive change that inhibits ROS-mediated cell senescence and participates in the maintenance of ROS-associated tumor-promoting mechanisms. Accordingly, targeting MTH1 in RAS-transformed tumor cells will not only induce proliferative defects but also potentially enhance therapeutic cytotoxicity by shifting cellular response away from pro-survival mechanisms.

Flavopiridol Synergizes with Sorafenib to Induce Cytotoxicity and Potentiate Antitumorigenic Activity in EGFR/HER-2 and Mutant RAS/RAF Breast Cancer Model Systems

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Teddy S Nagaria

2013-08-01

Full Text Available Oncogenic receptor tyrosine kinase (RTK signaling through the Ras-Raf-Mek-Erk (Ras-MAPK pathway is implicated in a wide array of carcinomas, including those of the breast. The cyclin-dependent kinases (CDKs are implicated in regulating proliferative and survival signaling downstream of this pathway. Here, we show that CDK inhibitors exhibit an order of magnitude greater cytotoxic potency than a suite of inhibitors targeting RTK and Ras-MAPK signaling in cell lines representative of clinically recognized breast cancer (BC subtypes. Drug combination studies show that the pan-CDK inhibitor, flavopiridol (FPD, synergistically potentiated cytotoxicity induced by the Raf inhibitor, sorafenib (SFN. This synergy was most pronounced at sub-EC50 SFN concentrations in MDA-MB-231 (KRAS-G13D and BRAF-G464V mutations, MDA-MB-468 [epidermal growth factor receptor (EGFR overexpression], and SKBR3 [ErbB2/EGFR2 (HER-2 overexpression] cells but not in hormone-dependent MCF-7 and T47D cells. Potentiation of SFN cytotoxicity by FPD correlated with enhanced apoptosis, suppression of retinoblastoma (Rb signaling, and reduced Mcl-1 expression. SFN and FPD were also tested in an MDA-MB-231 mammary fat pad engraftment model of tumorigenesis. Mice treated with both drugs exhibited reduced primary tumor growth rates and metastatic tumor load in the lungs compared to treatment with either drug alone, and this correlated with greater reductions in Rb signaling and Mcl-1 expression in resected tumors. These findings support the development of CDK and Raf co-targeting strategies in EGFR/HER-2-overexpressing or RAS/RAF mutant BCs.

p53 and ATF4 mediate distinct and additive pathways to skeletal muscle atrophy during limb immobilization

Science.gov (United States)

Fox, Daniel K.; Ebert, Scott M.; Bongers, Kale S.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.; Kunkel, Steven D.

2014-01-01

Immobilization causes skeletal muscle atrophy via complex signaling pathways that are not well understood. To better understand these pathways, we investigated the roles of p53 and ATF4, two transcription factors that mediate adaptations to a variety of cellular stresses. Using mouse models, we demonstrate that 3 days of muscle immobilization induces muscle atrophy and increases expression of p53 and ATF4. Furthermore, muscle fibers lacking p53 or ATF4 are partially resistant to immobilization-induced muscle atrophy, and forced expression of p53 or ATF4 induces muscle fiber atrophy in the absence of immobilization. Importantly, however, p53 and ATF4 do not require each other to promote atrophy, and coexpression of p53 and ATF4 induces more atrophy than either transcription factor alone. Moreover, muscle fibers lacking both p53 and ATF4 are more resistant to immobilization-induced atrophy than fibers lacking only p53 or ATF4. Interestingly, the independent and additive nature of the p53 and ATF4 pathways allows for combinatorial control of at least one downstream effector, p21. Using genome-wide mRNA expression arrays, we identified p21 mRNA as a skeletal muscle transcript that is highly induced in immobilized muscle via the combined actions of p53 and ATF4. Additionally, in mouse muscle, p21 induces atrophy in a manner that does not require immobilization, p53 or ATF4, and p21 is required for atrophy induced by immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as essential and complementary mediators of immobilization-induced muscle atrophy and discover p21 as a critical downstream effector of the p53 and ATF4 pathways. PMID:24895282

Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

Science.gov (United States)

Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

2015-01-01

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

RAS Insight

Science.gov (United States)

David Heimbrook, now CEO of the Frederick National Laboratory for Cancer Research, played a major role in a large pharma as it tried to develop an anti-RAS drug. Lessons from that failure inform the RAS Initiative today.

Active Erk Regulates Microtubule Stability in H-ras-Transformed Cells

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Rene E. Harrison

2001-01-01

Full Text Available Increasing evidence suggests that activated erk regulates cell functions, at least in part, by mechanisms that do not require gene transcription. Here we show that the map kinase, erk, decorates microtubules (MTs and mitotic spindles in both parental and mutant active rastransfected 10T1 /2 fibroblasts and MCF10A breast epithelial cells. Approximately 20% of total cellular erk decorated MTs in both cell lines. A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Activation of erk by the ras pathway coincided with a decrease in the stability of MT, as detected by a stability marker. The MKK1 inhibitor, PD98059 and transfection of a dominant negative MKK1 blocked ras-induced instability of MTs but did not modify the association of erk with MTs or affect MT stability of the parental cells. These results indicate that the subset of active erk kinase that associates with MTs contributes to their instability in the presence of a mutant active ras. The MT-associated subset of active erk likely contributes to the enhanced invasive and proliferative abilities of cells containing mutant active H-ras.

Human hypervariable sequences in risk assessment: rare Ha-ras alleles in cancer patients

International Nuclear Information System (INIS)

Krontiris, T.G.; DiMartino, N.A.; Mitcheson, H.D.; Lonergan, J.A.; Begg, C.; Parkinson, D.R.

1987-01-01

A variable tandem repeat (VTR) is responsible for the hyperallelism one kilobase 3' to the human c-Ha-ras-1 (Ha-ras) gene. Thirty-two distinct restriction fragments, comprising 3 allelic classes by frequency of occurrence, have thus far been detected in a sample size of approximately 800 caucasians. Rare Ha-ras alleles, 21 in all, are almost exclusively confined to the genomes of cancer patients. From their data the authors have computed the relative cancer risk associated with possession of a rare Ha-ras allele to be 27. To understand the molecular basis for this phenomenon, they have begun to clone Ha-ras fragments from nontumor DNA of cancer patients. They report here the weak activation, as detected by transfection and transformation of NIH 3T3 mouse cells, of two Ha-ras genes which were obtained from lymphocyte DNA of a melanoma patient. They have mapped the regions that confer this transforming activity to the fragment containing the VTR in one Ha-ras clone and the fragment containing gene coding sequences in the other

Zonation of heme synthesis enzymes in mouse liver and their regulation by β-catenin and Ha-ras.

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Braeuning, Albert; Schwarz, Michael

2010-11-01

Cytochrome P450 (CYP) hemoproteins play an important role in hepatic biotransformation. Recently, β-catenin and Ha-ras signaling have been identified as players controlling transcription of various CYP genes in mouse liver. The aim of the present study was to analyze the role of β-catenin and Ha-ras in the regulation of heme synthesis. Heme synthesis-related gene expression was analyzed in normal liver, in transgenic mice expressing activated β-catenin or Ha-ras, and in hepatomas. Regulation of the aminolevulinate dehydratase promoter was studied in vitro. Elevated expression of mRNAs and proteins involved in heme biosynthesis was linked to β-catenin activation in perivenous hepatocytes, in transgenic hepatocytes, and in hepatocellular tumors. Stimulation of the aminolevulinate dehydratase promoter by β-catenin was independent of the β-catenin/T-cell-specific transcription factor dimer. By contrast, activation of Ha-ras repressed heme synthesis-related gene expression. The present data suggest that β-catenin enhances the expression of both CYPs and heme synthesis-related genes, thus coordinating the availability of CYP apoprotein and its prosthetic group heme. The reciprocal regulation of heme synthesis by β-catenin and Ha-ras-dependent signaling supports our previous hypothesis that antagonistic action of these pathways plays a major role in the control of zonal gene expression in healthy mouse liver and aberrant expression patterns in hepatocellular tumors.

Lipoprotein-biomimetic nanostructure enables efficient targeting delivery of siRNA to Ras-activated glioblastoma cells via macropinocytosis

Science.gov (United States)

Huang, Jia-Lin; Jiang, Gan; Song, Qing-Xiang; Gu, Xiao; Hu, Meng; Wang, Xiao-Lin; Song, Hua-Hua; Chen, Le-Pei; Lin, Ying-Ying; Jiang, Di; Chen, Jun; Feng, Jun-Feng; Qiu, Yong-Ming; Jiang, Ji-Yao; Jiang, Xin-Guo; Chen, Hong-Zhuan; Gao, Xiao-Ling

2017-05-01

Hyperactivated Ras regulates many oncogenic pathways in several malignant human cancers including glioblastoma and it is an attractive target for cancer therapies. Ras activation in cancer cells drives protein internalization via macropinocytosis as a key nutrient-gaining process. By utilizing this unique endocytosis pathway, here we create a biologically inspired nanostructure that can induce cancer cells to `drink drugs' for targeting activating transcription factor-5 (ATF5), an overexpressed anti-apoptotic transcription factor in glioblastoma. Apolipoprotein E3-reconstituted high-density lipoprotein is used to encapsulate the siRNA-loaded calcium phosphate core and facilitate it to penetrate the blood-brain barrier, thus targeting the glioblastoma cells in a macropinocytosis-dependent manner. The nanostructure carrying ATF5 siRNA exerts remarkable RNA-interfering efficiency, increases glioblastoma cell apoptosis and inhibits tumour cell growth both in vitro and in xenograft tumour models. This strategy of targeting the macropinocytosis caused by Ras activation provides a nanoparticle-based approach for precision therapy in glioblastoma and other Ras-activated cancers.

A Fast, Sensitive and Accurate High Resolution Melting (HRM Technology-Based Assay to Screen for Common K-ras Mutations

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D. Kramer

2009-01-01

Full Text Available Background: Increasing evidence points to a negative correlation between K-ras mutations and patient’s response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed.

H-Ras and K-Ras Oncoproteins Induce Different Tumor Spectra When Driven by the Same Regulatory Sequences.

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Drosten, Matthias; Simón-Carrasco, Lucía; Hernández-Porras, Isabel; Lechuga, Carmen G; Blasco, María T; Jacob, Harrys K C; Fabbiano, Salvatore; Potenza, Nicoletta; Bustelo, Xosé R; Guerra, Carmen; Barbacid, Mariano

2017-02-01

Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-Ras G12V oncogene sequences. Germline expression of H-Ras G12V or K-Ras G12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-Ras G12V elicited papillomas and hematopoietic tumors, K-Ras G12V induced lung tumors and gastric lesions. Pulmonary expression of H-Ras G12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-Ras G12V but not K-Ras G12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-Ras G12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707-18. ©2016 AACR. ©2016 American Association for Cancer Research.

The Ras suppressor-1 (RSU-1 in cancer

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Lefteris C Zacharia

2017-04-01

Full Text Available Primary tumors are seldom the cause of death for cancer patients as most patients die from metastatic disease. Thus, deciphering metastatic mechanisms and key molecules involved is of utmost importance for the improved survival of cancer patients. Metastasis is a complex process in which cancer cells dissociate from the original tumor and spread to distant sites of the body. During the metastatic process, cancer cells lose contact both with the extracellular matrix (ECM and the neighboring cells within the primary tumor, thus invading though surrounding tissues. Therefore, ECM, and ECM-related adhesion proteins play a critical role in the metastatic process. Ras suppressor-1 (RSU-1 was first identified as a suppressor of Ras-dependent oncogenic transformation and is localized to cell-ECM adhesions where it is known to interact with the pro-survival adhesion protein PINCH-1. Although the connection to cancer is obvious, little is known regarding its expression in various cancer types. This opinion piece is focusing on recent literature regarding the expression of RSU-1 in various cancer types and the possible molecular mechanism of its action, pointing towards questions that need still to be addressed in this research field.

Mechanisms of membrane binding of small GTPase K-Ras4B farnesylated hypervariable region.

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Jang, Hyunbum; Abraham, Sherwin J; Chavan, Tanmay S; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I; Nussinov, Ruth; Gaponenko, Vadim

2015-04-10

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Myostatin regulates miR-431 expression via the Ras-Mek-Erk signaling pathway.

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Wu, Rimao; Li, Hu; Li, Tingting; Zhang, Yong; Zhu, Dahai

2015-05-29

MicroRNAs (miRNAs) play critical regulatory roles in controlling myogenic development both in vitro and in vivo; however, the molecular mechanisms underlying transcriptional regulation of miRNA genes in skeletal muscle cells are largely unknown. Here, using a microarray hybridization approach, we identified myostatin-regulated miRNA genes in skeletal muscle tissues by systematically searching miRNAs that are differentially expressed between wild-type and myostatin-null mice during development. We found that 116 miRNA genes were differentially expressed in muscles between these mice across different developmental stages. We further characterized myostatin-regulated miR-431 was upregulated in skeletal muscle tissues of myostatin-null mice. In functional studies, we found that overexpression of miR-431 in C2C12 myoblast cells attenuated myostatin-induced suppression of myogenic differentiation. Mechanistic studies further demonstrated that myostatin acted through the Ras-Mek-Erk signaling pathway to transcriptionally regulate miR-431 expression C2C12 cells. Our findings provide new insight into the mechanisms underlying transcriptional regulation of miRNA genes by myostatin during skeletal muscle development. Copyright © 2015 Elsevier Inc. All rights reserved.

RAS - Target Identification - Informatics

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The RAS Informatics lab group develops tools to track and analyze “big data” from the RAS Initiative, as well as analyzes data from external projects. By integrating internal and external data, this group helps improve understanding of RAS-driven cancers.

A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC).

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Yamamoto, Sayaka; Yako, Yuta; Fujioka, Yoichiro; Kajita, Mihoko; Kameyama, Takeshi; Kon, Shunsuke; Ishikawa, Susumu; Ohba, Yusuke; Ohno, Yusuke; Kihara, Akio; Fujita, Yasuyuki

2016-02-01

At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells. © 2016 Yamamoto, Yako, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Inactivation of the Fanconi anemia/BRCA pathway in lung and oral cancers: implications for treatment and survival.

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Marsit, Carmen J; Liu, Mei; Nelson, Heather H; Posner, Marshall; Suzuki, Makoto; Kelsey, Karl T

2004-01-29

Inactivation of the FANC-BRCA pathway via promoter methylation of the FANCF gene renders cells sensitive to DNA crosslinking agents, and has been identified in ovarian cancer cell lines and sporadic primary tumor tissues. We investigated epigenetic alterations in the FANC-BRCA pathway in head and neck squamous cell carcinomas (HNSCC) and non-small-cell lung cancers (NSCLC) using methylation-specific PCR. Promoter methylation of FANCF occurred in 15% (13/89) of HNSCCs and 14% (22/158) of NSCLCs. Methylation of BRCA1 occurred only in 6/158 NSCLC, and was limited to adenocarcinomas and large-cell carcinomas of the lung. No methylation of BRCA2 was detected. FANCF methylation was associated with a shorter duration of tobacco use (P=0.03) and a younger age of starting smoking (P=0.06) in NSCLC, and with a greater number of years of alcohol drinking (P=0.02) in HNSCC. In adenocarcinomas of the lung, FANCF promoter methylation was a significant predictor of poor survival with a hazard ratio of 3.1 (95% CI 1.2-7.9). This study demonstrates that inactivation of the FANC-BRCA pathway is relatively common in solid tumors and may be related to tobacco and alcohol exposure and survival of these patients.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

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Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Correlation between expression of p53, p21/WAF1, and MDM2 proteins and their prognostic significance in primary hepatocellular carcinoma

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Fu Jia

2009-12-01

Full Text Available Abstract Background Tumor Protein p53 (p53, cyclin-dependent kinase inhibitor 1A (p21/WAF1, and murine double minute 2 (MDM2 participate in the regulation of cell growth. Altered expression of these gene products has been found in malignant tumors and has been associated with poor prognosis. Our aim was to investigate the expression of the 3 proteins in hepatocellular carcinoma (HCC and their prognostic significance. Methods We examined p53, p21/WAF1, and MDM2 expression in 181 pairs of HCC tissues and the adjacent hepatic tissues by performing immunohistochemistry and examined the expression of the 3 proteins in 7 pairs of HCC tissues and the adjacent hepatic tissues by using western blot analysis. Results The expression of p53, p21/WAF1, and MDM2 in the HCC tissues was significantly higher than those in the adjacent hepatic tissues (P P = 0.008. A statistical correlation was observed between expression of p53 and p21/WAF1 (R = 0.380, P = 0.000, p53 and MDM2 (R = 0.299, P = 0.000, p21/WAF1 and MDM2 (R = 0.285, P = 0.000 in 181 liver tissues adjacent to the tumor. Patients with a low pathologic grade HCC (I+II had a higher tendency to express p53 on tumor cells than the patients with high pathologic grade HCC (III+IV (P = 0.007. Survival analysis showed that positive p21/WAF1 expression or/and negative MDM2 expression in HCC was a predictor of better survival of patients after tumor resection (P Conclusions The proteins p53, p21/WAF1, and MDM2 were overexpressed in all the HCC cases in this study, and p53 and p21/WAF1 overexpression were positively correlated. The expression of p21/WAF1 and MDM2 can be considered as 2 useful indicators for predicting the prognosis of HCC.

The S100P/RAGE signaling pathway regulates expression of microRNA-21 in colon cancer cells.

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Mercado-Pimentel, Melania E; Onyeagucha, Benjamin C; Li, Qing; Pimentel, Angel C; Jandova, Jana; Nelson, Mark A

2015-08-19

S100P signaling through the receptor for advanced glycation end-products (RAGE) contributes to colon cancer invasion and metastasis, but the mechanistic features of this process are obscure. Here, we investigate whether activation of S100P/RAGE signaling regulates oncogenic microRNA-21 (miR-21). We show that exogenous S100P up-regulates miR-21 levels in human colon cancer cells, whereas knockdown of S100P results in a decrease of miR-21. Furthermore, blockage of RAGE with anti-RAGE antibody suppresses S100P induction of miR-21. In addition, we found that S100P induction of miR-21 expression involves ERK and is suppressed by the MEK inhibitor U0126. Also, S100P treatment stimulates the enrichment of c-Fos, and AP-1 family members, at the miR-21 gene promoter. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

RAS Initiative - Events

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The NCI RAS Initiative has organized multiple events with outside experts to discuss how the latest scientific and technological breakthroughs can be applied to discover vulnerabilities in RAS-driven cancers.

Preoperative chemoradiation with capecitabine, irinotecan and cetuximab in rectal cancer: significance of pre-treatment and post-resection RAS mutations.

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Gollins, Simon; West, Nick; Sebag-Montefiore, David; Myint, Arthur Sun; Saunders, Mark; Susnerwala, Shabbir; Quirke, Phil; Essapen, Sharadah; Samuel, Leslie; Sizer, Bruce; Worlding, Jane; Southward, Katie; Hemmings, Gemma; Tinkler-Hundal, Emma; Taylor, Morag; Bottomley, Daniel; Chambers, Philip; Lawrie, Emma; Lopes, Andre; Beare, Sandy

2017-10-24

The influence of EGFR pathway mutations on cetuximab-containing rectal cancer preoperative chemoradiation (CRT) is uncertain. In a prospective phase II trial (EXCITE), patients with magnetic resonance imaging (MRI)-defined non-metastatic rectal adenocarinoma threatening/involving the surgical resection plane received pelvic radiotherapy with concurrent capecitabine, irinotecan and cetuximab. Resection was recommended 8 weeks later. The primary endpoint was histopathologically clear (R0) resection margin. Pre-planned retrospective DNA pyrosequencing (PS) and next generation sequencing (NGS) of KRAS, NRAS, PIK3CA and BRAF was performed on the pre-treatment biopsy and resected specimen. Eighty-two patients were recruited and 76 underwent surgery, with R0 resection in 67 (82%, 90%CI: 73-88%) (four patients with clinical complete response declined surgery). Twenty-four patients (30%) had an excellent clinical or pathological response (ECPR). Using NGS 24 (46%) of 52 matched biopsies/resections were discrepant: ten patients (19%) gained 13 new resection mutations compared to biopsy (12 KRAS, one PIK3CA) and 18 (35%) lost 22 mutations (15 KRAS, 7 PIK3CA). Tumours only ever testing RAS wild-type had significantly greater ECPR than tumours with either biopsy or resection RAS mutations (14/29 [48%] vs 10/51 [20%], P=0.008), with a trend towards increased overall survival (HR 0.23, 95% CI 0.05-1.03, P=0.055). This regimen was feasible and the primary study endpoint was met. For the first time using pre-operative rectal CRT, emergence of clinically important new resection mutations is described, likely reflecting intratumoural heterogeneity manifesting either as treatment-driven selective clonal expansion or a geographical biopsy sampling miss.

Carprofen Induction of p75NTR Dependent Apoptosis via the p38 MAPK Pathway in Prostate Cancer Cells

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Khwaja, Fatima S.; Quann, Emily J.; Pattabiraman, Nagarajan; Wynne, Shehla; Djakiew, Daniel

2008-01-01

The p75NTR functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we demonstrated that treatment with R-flurbiprofen or ibuprofen induced p75NTR expression in several prostate cancer cell lines leading to p75NTR mediated decreased survival. Utilizing the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico data base of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75NTR levels and inhibition of cell survival. Prostate (PC-3, DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75NTR associated loss of survival than breast (MCF7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant negative form of p75NTR prior to carprofen treatment partially rescued cell survival demonstrating a cause and effect relationship between carprofen induction of p75NTR levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF7 and 3T3 cells. Furthermore, siRNA knockdown of the p38 MAPK protein prevented induction of p75NTR by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 minute. Expression of a dominant negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75NTR protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75NTR dependent apoptosis via the p38 MAPK pathway in prostate cancer cells. PMID:18974393

Possible involvement of MSX-2 homeoprotein in v-ras-induced transformation.

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Takahashi, C; Akiyama, N; Kitayama, H; Takai, S; Noda, M

1997-04-01

A truncated MSX-2 homeoprotein was found to induce flat reversion when expressed in v-Ki-ras-transformed NIH3T3 cells. Although the expression of endogenous MSX-2 gene is low in most of the normal adult tissues examined, it is frequently activated in carcinoma-derived cell lines. Likewise, the gene is inactive in untransformed cells but is transcriptionally activated after transformation by v-Ki-ras oncogene, suggesting that the intact MSX-2 may play a positive, rather than suppressive, role in cell transformation. To test this possibility, we isolated a full-length human MSX-2 cDNA and tested its activities in two cell systems: fibroblast and myoblast. In NIH3T3 fibroblasts, although the gene by itself failed to confer a transformed phenotype, antisense MSX-2 cDNA as well as truncated MSX-2 cDNA interfered with the transforming activities of both v-Ki-ras and v-raf oncogene. In C2C12 myoblasts, MSX-2 was found to suppress MyoD gene expression, as do activated ras oncogenes, under certain culture conditions, and truncated MSX-2 cDNA was found to inhibit the activities of both MSX-2 and ras in this system as well. Our findings not only suggest that the truncated version MSX-2 may act as a dominant suppressor of intact MSX-2 but also raise the possibility that MSX-2 gene may be an important downstream target for the Ras signaling pathways.

Identification of H-Ras-Specific Motif for the Activation of Invasive Signaling Program in Human Breast Epithelial Cells

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Hae-Young Yong

2011-02-01

Full Text Available Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR, consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

Depressive symptoms predict head and neck cancer survival: Examining plausible behavioral and biological pathways.

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Zimmaro, Lauren A; Sephton, Sandra E; Siwik, Chelsea J; Phillips, Kala M; Rebholz, Whitney N; Kraemer, Helena C; Giese-Davis, Janine; Wilson, Liz; Bumpous, Jeffrey M; Cash, Elizabeth D

2018-03-01

Head and neck cancers are associated with high rates of depression, which may increase the risk for poorer immediate and long-term outcomes. Here it was hypothesized that greater depressive symptoms would predict earlier mortality, and behavioral (treatment interruption) and biological (treatment response) mediators were examined. Patients (n = 134) reported depressive symptomatology at treatment planning. Clinical data were reviewed at the 2-year follow-up. Greater depressive symptoms were associated with significantly shorter survival (hazard ratio, 0.868; 95% confidence interval [CI], 0.819-0.921; P ratio, 0.865; 95% CI, 0.774-0.966; P = .010), and poorer treatment response (odds ratio, 0.879; 95% CI, 0.803-0.963; P = .005). The poorer treatment response partially explained the depression-survival relation. Other known prognostic indicators did not challenge these results. Depressive symptoms at the time of treatment planning predict overall 2-year mortality. Effects are partly influenced by the treatment response. Depression screening and intervention may be beneficial. Future studies should examine parallel biological pathways linking depression to cancer survival, including endocrine disruption and inflammation. Cancer 2018;124:1053-60. © 2018 American Cancer Society. © 2018 American Cancer Society.

Absence of mutations in the coding sequence of the potential tumor suppressor 3pK in metastatic melanoma

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Houben Roland

2005-12-01

Full Text Available Abstract Background Activation of Ras or Raf contributes to tumorigenesis of melanoma. However, constitutive Raf activation is also a characteristic of the majority of benign melanocytic nevi and high intensity signaling of either Ras or Raf was found to induce growth inhibition and senescence rather than transformation. Since the chromosome 3p kinase (3pK is a target of the Ras/Raf/Mek/Erk signaling pathway which antagonizes the function of the oncogene and anti-differentiation factor Bmi-1, 3pK may function as a tumor suppressor in tumors with constitutive Ras/Raf activation. Consequently, we tested whether inactivating 3pK mutations are present in melanoma. Methods 30 metastatic melanoma samples, which were positive for activating mutations of either BRaf or NRas, were analyzed for possible mutations in the 3pk gene. The 10 coding exons and their flanking intron sequences were amplified by PCR and direct sequencing of the PCR products was performed. Results This analysis revealed that besides the presence of some single nucleotide polymorphisms in the 3pk gene, we could not detect any possible loss of function mutation in any of these 30 metastatic melanoma samples selected for the presence of activating mutations within the Ras/Raf/Mek/Erk signaling pathway. Conclusion Hence, in melanoma with constitutively active Ras/Raf inactivating mutations within the 3pk gene do not contribute to the oncogenic phenotype of this highly malignant tumor.

Simvastatin attenuates acrolein-induced mucin production in rats: involvement of the Ras/extracellular signal-regulated kinase pathway.

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Chen, Ya-Juan; Chen, Peng; Wang, Hai-Xia; Wang, Tao; Chen, Lei; Wang, Xun; Sun, Bei-Bei; Liu, Dai-Shun; Xu, Dan; An, Jing; Wen, Fu-Qiang

2010-06-01

Airway mucus overproduction is a cardinal feature of airway inflammatory diseases, such as chronic obstructive pulmonary disease and cystic fibrosis. Since the small G-protein Ras is known to modulate cellular functions in the lung, we sought to investigate whether the Ras inhibitor simvastatin could attenuate acrolein-induced mucin production in rat airways. Rats were exposed to acrolein for 12 days, after first being pretreated intragastrically for 24 h with either simvastatin alone or simvastatin in combination with mevalonate, which prevents the isoprenylation needed for Ras activation. Lung tissue was analyzed for extracellular signal-regulated kinase (ERK) activity, goblet cell metaplasia and mucin production. To analyze the effect of simvastatin on mucin production in more detail, acrolein-exposed human airway epithelial NCI-H292 cells were pretreated with simvastatin alone or together with mevalonate. Culture medium was collected to detect mucin secretion, and cell lysates were examined for Ras-GTPase activity and epidermal growth factor receptor (EGFR)/ERK phosphorylation. In vivo, simvastatin treatment dose-dependently suppressed acrolein-induced goblet cell hyperplasia and metaplasia in bronchial epithelium and inhibited ERK phosphorylation in rat lung homogenates. Moreover, simvastatin inhibited Muc5AC mucin synthesis at both the mRNA and protein levels in the lung. In vitro, simvastatin pretreatment attenuated the acrolein-induced significant increase in MUC5AC mucin expression, Ras-GTPase activity and EGFR/ERK phosphorylation. These inhibitory effects of simvastatin were neutralized by mevalonate administration both in vitro and in vivo. Our results suggest that simvastatin may attenuate acrolein-induced mucin protein synthesis in the airway and airway inflammation, possibly by blocking ERK activation mediated by Ras protein isoprenylation. Thus, the evidence from the experiment suggests that human trials are warranted to determine the potential

Fetisisme Ras Kaukasoid dan Ras Mongoloid Sebagai Strategi Pemasaran dalam Sinetron Indonesia

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Dimas Yudhistira

2014-12-01

ABSTRAK   Budaya populer yang tumbuh seiring dengan industrialisasi memengaruhi produksi per- filman di Indonesia. Salah satu genre perfilman di Indonesia adalah sinetron. Sinetron yang di- kategorikan sebagai produk seni kitsch memiliki dua kriteria yaitu sebagai komoditi seni yang populer dan sebagai komoditi dagang yang menghasilkan keuntungan ekonomis. Sebagai se- buah produk seni kitsch yang merupakan dasar pembuatan karyanya adalah selera masyarakat kebanyakan maka sinetron harus jeli dalam melihat keadaan dan latar belakang masyarakat. Penelitian ini menggunakan metode kualitatif. Hasil penelitian ini menggambarkan masyara- kat Indonesia yang merupakan ras Melayu telah dijajah oleh ras Kaukasoid dan Mongoloid sebelum tahun 1945 dan setelahnya. Efek dari penjajahan ini adalah ras Melayu telah ditanami fantasi yang menjadi stereotip mengenai ras Kaukasoid dan Mongoloid yang berakhir dengan fetisisme. Fetisisme ini dijadikan sebagai strategi pemasaran oleh produser dan sutradara un- tuk menarik antusiasme calon penonton sinetron. Caranya dengan menampilkan aktor dan aktris Melayu keturunan Kaukasoid dan Mongoloid sebagai pemeran utama.   Kata kunci: sinetron, seni kitsch, ras, fetisisme

A Novel Ras Inhibitor (MDC-1016 Reduces Human Pancreatic Tumor Growth in Mice

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Gerardo G Mackenzie

2013-10-01

Full Text Available Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its persistent resistance to chemotherapy. The currently limited treatment options for pancreatic cancer underscore the need for more efficient agents. Because activating Kras mutations initiate and maintain pancreatic cancer, inhibition of this pathway should have a major therapeutic impact. We synthesized phospho-farnesylthiosalicylic acid (PFTS; MDC-1016 and evaluated its efficacy, safety, and metabolism in preclinical models of pancreatic cancer. PFTS inhibited the growth of human pancreatic cancer cells in culture in a concentration- and time-dependent manner. In an MIA PaCa-2 xenograft mouse model, PFTS at a dose of 50 and 100 mg/kg significantly reduced tumor growth by 62% and 65% (P < .05 vs vehicle control. Furthermore, PFTS prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. PFTS appeared to be safe, with the animals showing no signs of toxicity during treatment. Following oral administration, PFTS was rapidly absorbed, metabolized to FTS and FTS glucuronide, and distributed through the blood to body organs. Mechanistically, PFTS inhibited Ras-GTP, the active form of Ras, both in vitro and in vivo, leading to the inhibition of downstream effector pathways c-RAF/mitogen-activated protein-extracellular signal-regulated kinase (ERK kinase (MEK/ERK1/2 kinase and phosphatidylinositol 3-kinase/AKT. In addition, PFTS proved to be a strong combination partner with phospho-valproic acid, a novel signal transducer and activator of transcription 3 (STAT3 inhibitor, displaying synergy in the inhibition of pancreatic cancer growth. In conclusion, PFTS, a direct Ras inhibitor, is an efficacious agent for the treatment of pancreatic cancer in preclinical models, deserving further evaluation.

Pressure modulation of Ras-membrane interactions and intervesicle transfer.

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Kapoor, Shobhna; Werkmüller, Alexander; Goody, Roger S; Waldmann, Herbert; Winter, Roland

2013-04-24

Proteins attached to the plasma membrane frequently encounter mechanical stresses, including high hydrostatic pressure (HHP) stress. Signaling pathways involving membrane-associated small GTPases (e.g., Ras) have been identified as critical loci for pressure perturbation. However, the impact of mechanical stimuli on biological outputs is still largely terra incognita. The present study explores the effect of HHP on the membrane association, dissociation, and intervesicle transfer process of N-Ras by using a FRET-based assay to obtain the kinetic parameters and volumetric properties along the reaction path of these processes. Notably, membrane association is fostered upon pressurization. Conversely, depending on the nature and lateral organization of the lipid membrane, acceleration or retardation is observed for the dissociation step. In addition, HHP can be inferred as a positive regulator of N-Ras clustering, in particular in heterogeneous membranes. The susceptibility of membrane interaction to pressure raises the idea of a role of lipidated signaling molecules as mechanosensors, transducing mechanical stimuli to chemical signals by regulating their membrane binding and dissociation. Finally, our results provide first insights into the influence of pressure on membrane-associated Ras-controlled signaling events in organisms living under extreme environmental conditions such as those that are encountered in the deep sea and sub-seafloor environments, where pressures reach the kilobar (100 MPa) range.

Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells.

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Perros, M; Deleu, L; Vanacker, J M; Kherrouche, Z; Spruyt, N; Faisst, S; Rommelaere, J

1995-01-01

The activity of the P4 promoter of the parvovirus minute virus of mice (prototype strain MVMp) is stimulated in ras-transformed FREJ4 cells compared with the parental FR3T3 line. This activation may participate in the oncolytic effect of parvoviruses, given that P4 drives a transcriptional unit encoding cytotoxic nonstructural proteins. Our results suggest that the higher transcriptional activity of promoter P4 in FREJ4 cells is mediated at least in part by upstream CRE elements. Accordingly, mutations in the CRE motifs impair P4 function more strongly in the FREJ4 derivative than in its FR3T3 parent. Further evidence that these elements contribute to hyperactivity of the P4 promoter in the ras transformant is the fact that they form distinct complexes with proteins from FREJ4 and FR3T3 cell extracts. This difference can be abolished by treating the FREJ4 cell extracts with cyclic AMP-dependent protein kinase (PKA) or treating original cultures with a PKA activator. These findings can be linked with two previously reported features of ras-transformed cells: the activation of a PKA-inhibited protein kinase cascade and the reduction of PKA-induced protein phosphorylation. In keeping with these facts, P4-directed gene expression can be up- or downmodulated in vivo by exposing cells to known inhibitors or activators of PKA, respectively. PMID:7636996

p62 modulates Akt activity via association with PKCζ in neuronal survival and differentiation

International Nuclear Information System (INIS)

Joung, Insil; Kim, Hak Jae; Kwon, Yunhee Kim

2005-01-01

p62 is a ubiquitously expressed phosphoprotein that interacts with a number of signaling molecules and a major component of neurofibrillary tangles in the brain of Alzheimer's disease patients. It has been implicated in important cellular functions such as cell proliferation and anti-apoptotic pathways. In this study, we have addressed the potential role of p62 during neuronal differentiation and survival using HiB5, a rat neuronal progenitor cell. We generated a recombinant adenovirus encoding T7-epitope tagged p62 to reliably transfer p62 cDNA into the neuronal cells. The results show that an overexpression of p62 led not only to neuronal differentiation, but also to decreased cell death induced by serum withdrawal in HiB5 cells. In this process p62-dependent Akt phosphorylation occurred via the release of Akt from PKCζ by association of p62 and PKCζ, which is known as a negative regulator of Akt activation. These findings indicate that p62 facilitates cell survival through novel signaling cascades that result in Akt activation. Furthermore, we found that p62 expression was induced during neuronal differentiation. Taken together, the data suggest p62 is a regulator of neuronal cell survival and differentiation

Regional imbalanced activation of the calcineurin/BAD apoptotic pathway and the PI3K/Akt survival pathway after myocardial infarction.

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Li, Tieluo; Kilic, Ahmet; Wei, Xufeng; Wu, Changfu; Schwartzbauer, Gary; Yankey, G Kwame; DeFilippi, Christopher; Bond, Meredith; Wu, Zhongjun J; Griffith, Bartley P

2013-06-05

The underlying molecular mechanisms of the remodeling after myocardial infarction (MI) remain unclear. The purpose of this study was to investigate the role of a survival pathway (PI3K/Akt) and an apoptosis pathway (calcineurin/BAD) in the remodeling after MI in a large animal model. Ten Dorset hybrid sheep underwent 25% MI in the left ventricle (LV, n=10). Five sheep were used as sham control. The regional strain was calculated from sonomicrometry. Apoptosis and the activation of the PI3K/Akt and calcineurin/BAD pathways were evaluated in the non-ischemic adjacent zone and the remote zone relative to infarct by immunoblotting, immunoprecipitation, and immunofluorescence staining. Dilation and dysfunction of LV were present at 12 weeks after MI. The regional strain in the adjacent zone was significantly higher than in the remote zone at 12 weeks (36.6 ± 4.0% vs 9.5 ± 3.6%, pBAD pathways were activated in the adjacent zone. Dephosphorylation and translocation of BAD were evident in the adjacent zone. Regional correlation between the strain and the expression of calcineurin/BAD indicated that the activation was strain-related (R(2)=0.46, 0.48, 0.39 for calcineurin, BAD, mitochondrial BAD, respectively, pBAD apoptotic pathways were concomitantly activated in the non-ischemic adjacent zone after MI. The calcineurin/BAD pathway is strain related and its imbalanced activation may be one of the causes of progressive remodeling after MI. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory.

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Gyurkó, M Dávid; Csermely, Péter; Sőti, Csaba; Steták, Attila

2015-10-15

The Ras GTPase activating proteins (RasGAPs) are regulators of the conserved Ras/MAPK pathway. Various roles of some of the RasGAPs in learning and memory have been reported in different model systems, yet, there is no comprehensive study to characterize all gap genes in any organism. Here, using reverse genetics and neurobehavioural tests, we studied the role of all known genes of the rasgap family in C. elegans in associative learning and memory. We demonstrated that their proteins are implicated in different parts of the learning and memory processes. We show that gap-1 contribute redundantly with gap-3 to the chemosensation of volatile compounds, gap-1 plays a major role in associative learning, while gap-2 and gap-3 are predominantly required for short- and long-term associative memory. Our results also suggest that the C. elegans Ras orthologue let-60 is involved in multiple processes during learning and memory. Thus, we show that the different classes of RasGAP proteins are all involved in cognitive function and their complex interplay ensures the proper formation and storage of novel information in C. elegans.

miR-411-5p inhibits proliferation and metastasis of breast cancer cell via targeting GRB2

International Nuclear Information System (INIS)

Zhang, Yunda; Xu, Guoxing; Liu, Gang; Ye, Yongzhi; Zhang, Chuankai; Fan, Chuannan; Wang, Haibin; Cai, Huali; Xiao, Rui; Huang, Zhengjie; Luo, Qi

2016-01-01

miR-411-5p (previously called miR-411) is severely involved in human diseases, however, the relationship between miR-411-5p and breast cancer has not been investigated thoroughly. Here, we found that the expression of miR-411-5p was downregulated in breast cancer tissues compared with their matched adjacent non-neoplastic tissues. In addition, the expression of miR-411-5p was also lower in breast cancer cell lines in contrast with MCF-10A. Moreover, we investigated the target and mechanism of miR-411-5p in breast cancer using mimic and inhibitor, and demonstrated the involvement of GRB2 and Ras activation. Ectopic expression of miR-411-5p suppressed the breast cancer cell proliferation, migration and invasion while low expression of miR-411-5p exhibited the opposite effect. Furthermore, GRB2 was demonstrated to be significantly overexpressed in breast cancer tissues compared with normal tissues, and low expression of GRB2 had a longer overall survival compared with high expression of GRB2 in breast cancer. In general, our study shed light on the miR-411-5p related mechanism in the progression of breast cancer and, miR-411-5p/GRB2/Ras axis is potential to be molecular target for breast cancer therapy. - Highlights: • miR-411-5p is downregulated in breast cancer tissues and cell lines. • miR-411-5p inhibits breast cancer cells growth, migration and invasion in vitro. • GRB2 is a direct target of miR-411-5p in breast cancer. • GRB2 is overexpressed in breast cancer and associates with disease outcome. • miR-411-5p suppresses breast cancer progression though GRB2-SOS-Ras pathway.

miR-411-5p inhibits proliferation and metastasis of breast cancer cell via targeting GRB2

Energy Technology Data Exchange (ETDEWEB)

Zhang, Yunda [Department of Gastrointestinal Surgery, First Affiliated Hospital of Xiamen University, Xiamen 361003 (China); State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen 361102 (China); Xu, Guoxing [Department of Gastrointestinal Surgery, First Affiliated Hospital of Xiamen University, Xiamen 361003 (China); Department of Gastrointestinal Surgery, First Clinical Medical College of Fujian Medical University, Fuzhou 350005 (China); Liu, Gang; Ye, Yongzhi [Department of Gastrointestinal Surgery, First Affiliated Hospital of Xiamen University, Xiamen 361003 (China); Zhang, Chuankai [Department of Gastrointestinal Surgery, First Affiliated Hospital of Xiamen University, Xiamen 361003 (China); State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen 361102 (China); Fan, Chuannan [State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen 361102 (China); Wang, Haibin; Cai, Huali; Xiao, Rui [Department of Gastrointestinal Surgery, First Affiliated Hospital of Xiamen University, Xiamen 361003 (China); Department of Gastrointestinal Surgery, First Clinical Medical College of Fujian Medical University, Fuzhou 350005 (China); Huang, Zhengjie, E-mail: huangzhengjie@xmu.edu.cn [Department of Gastrointestinal Surgery, First Affiliated Hospital of Xiamen University, Xiamen 361003 (China); Department of Gastrointestinal Surgery, First Clinical Medical College of Fujian Medical University, Fuzhou 350005 (China); Luo, Qi, E-mail: luoqixmzsh@126.com [Department of Gastrointestinal Surgery, First Affiliated Hospital of Xiamen University, Xiamen 361003 (China); Department of Gastrointestinal Surgery, First Clinical Medical College of Fujian Medical University, Fuzhou 350005 (China)

2016-08-05

miR-411-5p (previously called miR-411) is severely involved in human diseases, however, the relationship between miR-411-5p and breast cancer has not been investigated thoroughly. Here, we found that the expression of miR-411-5p was downregulated in breast cancer tissues compared with their matched adjacent non-neoplastic tissues. In addition, the expression of miR-411-5p was also lower in breast cancer cell lines in contrast with MCF-10A. Moreover, we investigated the target and mechanism of miR-411-5p in breast cancer using mimic and inhibitor, and demonstrated the involvement of GRB2 and Ras activation. Ectopic expression of miR-411-5p suppressed the breast cancer cell proliferation, migration and invasion while low expression of miR-411-5p exhibited the opposite effect. Furthermore, GRB2 was demonstrated to be significantly overexpressed in breast cancer tissues compared with normal tissues, and low expression of GRB2 had a longer overall survival compared with high expression of GRB2 in breast cancer. In general, our study shed light on the miR-411-5p related mechanism in the progression of breast cancer and, miR-411-5p/GRB2/Ras axis is potential to be molecular target for breast cancer therapy. - Highlights: • miR-411-5p is downregulated in breast cancer tissues and cell lines. • miR-411-5p inhibits breast cancer cells growth, migration and invasion in vitro. • GRB2 is a direct target of miR-411-5p in breast cancer. • GRB2 is overexpressed in breast cancer and associates with disease outcome. • miR-411-5p suppresses breast cancer progression though GRB2-SOS-Ras pathway.

Specific repression of mutant K-RAS by 10-23 DNAzyme: Sensitizing cancer cell to anti-cancer therapies

International Nuclear Information System (INIS)

Yu, S.-H.; Wang, T.-H.; Au, L.-C.

2009-01-01

Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU → GUU) at the GU dinucleotide while left the wild-type (WT) K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.

Inactivation of CDK/pRb pathway normalizes survival pattern of lymphoblasts expressing the FTLD-progranulin mutation c.709-1G>A.

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Carolina Alquezar

Full Text Available BACKGROUND: Mutations in the progranulin (PGRN gene, leading to haploinsufficiency, cause familial frontotemporal lobar degeneration (FTLD-TDP, although the pathogenic mechanism of PGRN deficit is largely unknown. Allelic loss of PGRN was previously shown to increase the activity of cyclin-dependent kinase (CDK CDK6/pRb pathway in lymphoblasts expressing the c.709-1G>A PGRN mutation. Since members of the CDK family appear to play a role in neurodegenerative disorders and in apoptotic death of neurons subjected to various insults, we investigated the role of CDK6/pRb in cell survival/death mechanisms following serum deprivation. METHODOLOGY/PRINCIPAL FINDINGS: We performed a comparative study of cell viability after serum withdrawal of established lymphoblastoid cell lines from control and carriers of c.709-1G>A PGRN mutation, asymptomatic and FTLD-TDP diagnosed individuals. Our results suggest that the CDK6/pRb pathway is enhanced in the c.709-1G>A bearing lymphoblasts. Apparently, this feature allows PGRN-deficient cells to escape from serum withdrawal-induced apoptosis by decreasing the activity of executive caspases and lowering the dissipation of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. Inhibitors of CDK6 expression levels like sodium butyrate or the CDK6 activity such as PD332991 were able to restore the vulnerability of lymphoblasts from FTLD-TDP patients to trophic factor withdrawal. CONCLUSION/SIGNIFICANCE: The use of PGRN-deficient lymphoblasts from FTLD-TDP patients may be a useful model to investigate cell biochemical aspects of this disease. It is suggested that CDK6 could be potentially a therapeutic target for the treatment of the FTLD-TDP.

Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers

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Liu, Yang; Gao, Weimin; Siegfried, Jill M; Weissfeld, Joel L; Luketich, James D; Keohavong, Phouthone

2007-01-01

Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors. Promoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies. The RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene. Our results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of

Epac activation sensitizes rat sensory neurons via activation of Ras

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Shariati, Behzad; Thompson, Eric L.; Nicol, Grant D.; Vasko, Michael R.

2015-01-01

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2′-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. PMID:26596174

Epac activation sensitizes rat sensory neurons through activation of Ras.

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Shariati, Behzad; Thompson, Eric L; Nicol, Grant D; Vasko, Michael R

2016-01-01

Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. Copyright © 2015 Elsevier Inc. All rights reserved.

Induction of nonapoptotic cell death by activated Ras requires inverse regulation of Rac1 and Arf6.

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Bhanot, Haymanti; Young, Ashley M; Overmeyer, Jean H; Maltese, William A

2010-10-01

Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.

Effects of mutant human Ki-rasG12C gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

International Nuclear Information System (INIS)

Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.; Moore, Joseph E.; Mosley, Libyadda J.; D'Agostino, Ralph B.; Pettenati, Mark J.; Miller, Mark Steven

2008-01-01

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras G12C allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 μg/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras G12C allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 μg/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 μg/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 μg/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras G12C expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models

RB1CC1 activates RB1 pathway and inhibits proliferation and cologenic survival in human cancer.

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Tokuhiro Chano

2010-06-01

Full Text Available RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200 plays a role in the enhancement of the RB1 pathway through the direct binding to a GC-rich region 201bp upstream (from the initiation ATG of the RB1 promoter. Here, we identified hSNF5 and p53 as the binding partners of RB1CC1 by immunoprecipitation and immunofluorescence assays. Interaction between these molecules and the RB1 pathway was analyzed by the assays of chromatin immunoprecipitation, luciferase-reporter, reverse transcription-polymerase chain reaction and immunoblot. The tumor growth suppression by RB1CC1 was evaluated by flow cytometry or by a cell growth assay. The nuclear RB1CC1 complex involving hSNF5 and/or p53 activated transcription of RB1, p16 and p21, and suppressed tumor cell growth. Furthermore, nuclear RB1CC1 expression significantly correlated with those of RB1 and p16 in breast cancer tissue in vivo, and the Ki-67 proliferation index was dependent on p53 as well as RB1CC1. The present study indicates that RB1CC1 together with hSNF5 and/or p53 enhances the RB1 pathway through transcriptional activation of RB1, p16 and p21. Evaluation of RB1CC1 expression combined with RB1 and p53 status is expected to provide useful information in clinical practice and future therapeutic strategies in breast cancer.

A New Strategy to Control and Eradicate "Undruggable" Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology.

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Van Sciver, Robert E; Lee, Michael P; Lee, Caroline Dasom; Lafever, Alex C; Svyatova, Elizaveta; Kanda, Kevin; Colliver, Amber L; Siewertsz van Reesema, Lauren L; Tang-Tan, Angela M; Zheleva, Vasilena; Bwayi, Monicah N; Bian, Minglei; Schmidt, Rebecca L; Matrisian, Lynn M; Petersen, Gloria M; Tang, Amy H

2018-05-14

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely "undruggable". Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.

Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

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Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

2009-06-01

Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM. (c) 2009 Wiley-Liss, Inc.

Genetic variations in radiation and chemotherapy drug action pathways and survival in locoregionally advanced nasopharyngeal carcinoma treated with chemoradiotherapy.

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Huai Liu

Full Text Available BACKGROUND AND PURPOSE: Treatment outcomes vary greatly in patients with nasopharyngeal carcinoma (NPC. The purpose of this study is to evaluate the influence of radiation and chemotherapy drug action pathway gene polymorphisms on the survival of patients with locoregionally advanced NPC treated with cisplatin- and fluorouracil-based chemoradiotherapy. MATERIAL AND METHODS: Four hundred twenty-one consecutive patients with locoregionally advanced NPC were prospectively recruited. We utilized a pathway approach and examined 18 polymorphisms in 13 major genes. Polymorphisms were detected using the LDR-PCR technique. Multifactor dimensionality reduction (MDR analysis was performed to detect potential gene-gene interaction. RESULTS: After adjustment for clinicopathological characteristics, overall survival was significantly decreased in patients with the MPO rs2243828 CT/CC genotype (HR=2.453, 95% CI, 1.687-3.566, P<0.001. The ERCC1 rs3212986 CC (HR=1.711, 95% CI, 1.135-2.579, P=0.010, MDM2 rs2279744 GT/GG (HR=1.743, 95% CI, 1.086-2.798, P=0.021, MPO rs2243828 CT/CC (HR=3.184, 95% CI, 2.261-4.483, P<0.001 and ABCB1 rs2032582 AT/AA (HR=1.997, 95% CI, 1.086-3.670, P=0.026 genotypes were associated with poor progression-free survival. Prognostic score models based on independent prognostic factors successfully classified patients into low-, intermediate-, and high-risk groups. Furthermore, MDR analysis showed no significant interaction between polymorphisms. CONCLUSIONS: Four single nucleotide polymorphisms were associated with survival in patients with locoregionally advanced NPC treated with cisplatin- and fluorouracil-based chemoradiotherapy. Combining clinical prognostic factors with genetic information was valuable in identifying patients with different risk.

Epidermal Growth Factor Receptor and K-RAS status in two cohorts of squamous cell carcinomas

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Van Laethem Jean-Luc

2010-05-01

Full Text Available Abstract Background With the availability of effective anti-EGFR therapies for various solid malignancies, such as non-cell small lung cancer, colorectal cancer and squamous cell carcinoma of the head and neck, the knowledge of EGFR and K-RAS status becomes clinically important. The aim of this study was to analyse EGFR expression, EGFR gene copy number and EGFR and K-RAS mutations in two cohorts of squamous cell carcinomas, specifically anal canal and tonsil carcinomas. Methods Formalin fixed, paraffin-embedded tissues from anal and tonsil carcinoma were used. EGFR protein expression and EGFR gene copy number were analysed by means of immunohistochemistry and fluorescence in situ hybridisation. The somatic status of the EGFR gene was investigated by PCR using primers specific for exons 18 through 21. For the K-RAS gene, PCR was performed using exon 2 specific primers. Results EGFR immunoreactivity was present in 36/43 (83.7% of anal canal and in 20/24 (83.3% of tonsil squamous cell carcinomas. EGFR amplification was absent in anal canal tumours (0/23, but could be identified in 4 of 24 tonsil tumours. From 38 anal canal specimens, 26 specimens were successfully analysed for exon 18, 30 for exon 19, 34 for exon 20 and 30 for exon 21. No EGFR mutations were found in the investigated samples. Thirty samples were sequenced for K-RAS exon 2 and no mutation was identified. From 24 tonsil specimens, 22 were successfully analysed for exon 18 and all 24 specimens for exon 19, 20 and 21. No EGFR mutations were found. Twenty-two samples were sequenced for K-RAS exon 2 and one mutation c.53C > A was identified. Conclusion EGFR mutations were absent from squamous cell carcinoma of the anus and tonsils, but EGFR protein expression was detected in the majority of the cases. EGFR amplification was seen in tonsil but not in anal canal carcinomas. In our investigated panel, only one mutation in the K-RAS gene of a tonsil squamous cell carcinoma was identified

Epidermal Growth Factor Receptor and K-RAS status in two cohorts of squamous cell carcinomas

International Nuclear Information System (INIS)

Van Damme, Nancy; Pauwels, Patrick; Peeters, Marc; Deron, Philippe; Van Roy, Nadine; Demetter, Pieter; Bols, Alain; Dorpe, Jo Van; Baert, Filip; Van Laethem, Jean-Luc; Speleman, Franki

2010-01-01

With the availability of effective anti-EGFR therapies for various solid malignancies, such as non-cell small lung cancer, colorectal cancer and squamous cell carcinoma of the head and neck, the knowledge of EGFR and K-RAS status becomes clinically important. The aim of this study was to analyse EGFR expression, EGFR gene copy number and EGFR and K-RAS mutations in two cohorts of squamous cell carcinomas, specifically anal canal and tonsil carcinomas. Formalin fixed, paraffin-embedded tissues from anal and tonsil carcinoma were used. EGFR protein expression and EGFR gene copy number were analysed by means of immunohistochemistry and fluorescence in situ hybridisation. The somatic status of the EGFR gene was investigated by PCR using primers specific for exons 18 through 21. For the K-RAS gene, PCR was performed using exon 2 specific primers. EGFR immunoreactivity was present in 36/43 (83.7%) of anal canal and in 20/24 (83.3%) of tonsil squamous cell carcinomas. EGFR amplification was absent in anal canal tumours (0/23), but could be identified in 4 of 24 tonsil tumours. From 38 anal canal specimens, 26 specimens were successfully analysed for exon 18, 30 for exon 19, 34 for exon 20 and 30 for exon 21. No EGFR mutations were found in the investigated samples. Thirty samples were sequenced for K-RAS exon 2 and no mutation was identified. From 24 tonsil specimens, 22 were successfully analysed for exon 18 and all 24 specimens for exon 19, 20 and 21. No EGFR mutations were found. Twenty-two samples were sequenced for K-RAS exon 2 and one mutation c.53C > A was identified. EGFR mutations were absent from squamous cell carcinoma of the anus and tonsils, but EGFR protein expression was detected in the majority of the cases. EGFR amplification was seen in tonsil but not in anal canal carcinomas. In our investigated panel, only one mutation in the K-RAS gene of a tonsil squamous cell carcinoma was identified. This indicates that EGFR and K-RAS mutation analysis is not

Impaired binding of 14-3-3 to C-RAF in noonan syndrome suggests new approaches in diseases with increased ras signaling

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Molzan, M.; Schumacher, B.; Ottmann, C.; Baljuls, A.; Polzien, L.; Weyand, M.; Thiel, P.; Rose, R.; Rose, M.; Kuhenne, P.; Kaiser, M.; Rapp, U.R.; Kuhlmann, J.; Ottmann, C.

2010-01-01

The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser(259), a regulatory site for inhibition by 14-3-3 proteins. We

Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway

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D’Ambrosio, Steven M.; Han, Chunhua; Pan, Li; Kinghorn, A. Douglas; Ding, Haiming

2011-01-01

Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003), was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compound 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not compound 1, prevented EGF-induced activation of EGFR (Tyr1173). When compounds 1 and 2 were combined they synergistically inhibited c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation, and human oral cancer cell proliferation. The present data suggest that the potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins that target two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. PMID:21596018

About the RAS Initiative

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The RAS Initiative, a "hub and spoke" model, connects researchers to better understand and target the more than 30% of cancers driven by mutations in RAS genes. Includes oversight and contact information.

Long Non-Coding RNAs Embedded in the Rb and p53 Pathways

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Subramanian, Murugan; Jones, Matthew F.; Lal, Ashish, E-mail: ashish.lal@nih.gov [Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

2013-12-04

In recent years, long non-coding RNAs (lncRNAs) have gained significant attention as a novel class of gene regulators. Although a small number of lncRNAs have been shown to regulate gene expression through diverse mechanisms including transcriptional regulation, mRNA splicing and translation, the physiological function and mechanism of action of the vast majority are not known. Profiling studies in cell lines and tumor samples have suggested a potential role of lncRNAs in cancer. Indeed, distinct lncRNAs have been shown to be embedded in the p53 and Rb networks, two of the major tumor suppressor pathways that control cell cycle progression and survival. Given the fact that inactivation of Rb and p53 is a hallmark of human cancer, in this review we discuss recent evidence on the function of lncRNAs in the Rb and p53 signaling pathways.

Long Non-Coding RNAs Embedded in the Rb and p53 Pathways

International Nuclear Information System (INIS)

Subramanian, Murugan; Jones, Matthew F.; Lal, Ashish

2013-01-01

In recent years, long non-coding RNAs (lncRNAs) have gained significant attention as a novel class of gene regulators. Although a small number of lncRNAs have been shown to regulate gene expression through diverse mechanisms including transcriptional regulation, mRNA splicing and translation, the physiological function and mechanism of action of the vast majority are not known. Profiling studies in cell lines and tumor samples have suggested a potential role of lncRNAs in cancer. Indeed, distinct lncRNAs have been shown to be embedded in the p53 and Rb networks, two of the major tumor suppressor pathways that control cell cycle progression and survival. Given the fact that inactivation of Rb and p53 is a hallmark of human cancer, in this review we discuss recent evidence on the function of lncRNAs in the Rb and p53 signaling pathways

Galectin-3 mediates cross-talk between K-Ras and Let-7c tumor suppressor microRNA.

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Ran Levy

Full Text Available BACKGROUND: Galectin-3 (Gal-3 and active (GTP-bound K-Ras contribute to the malignant phenotype of many human tumors by increasing the rate of cell proliferation, survival, and migration. These Gal-3-mediated effects result from a selective binding to K-Ras.GTP, causing increased nanoclustering in the cell membrane and leading to robust Ras signaling. Regulation of the interactions between Gal-3 and active K-Ras is not fully understood. METHODS AND FINDINGS: To gain a better understanding of what regulates the critical interactions between these two proteins, we examined the role of Gal-3 in the regulation of K-Ras by using Gal-3-knockout mouse embryonic-fibroblasts (Gal-3-/- MEFs and/or Gal-3/Gal-1 double-knockout MEFs. We found that knockout of Gal-3 induced strong downregulation (∼60% of K-Ras and K-Ras.GTP. The downregulation was somewhat more marked in the double-knockout MEFs, in which we also detected robust inhibition(∼50% of ERK and Akt activation. These additional effects are probably attributable to inhibition of the weak interactions of K-Ras.GTP with Gal-1. Re-expression of Gal-3 reversed the phenotype of the Gal-3-/- MEFs and dramatically reduced the disappearance of K-Ras in the presence of cycloheximide to the levels seen in wild-type MEFs. Furthermore, phosphorylation of Gal-3 by casein kinase-1 (CK-1 induced translocation of Gal-3 from the nucleus to the cytoplasm and the plasma membrane, leading to K-Ras stabilization accompanied by downregulation of the tumor suppressor miRNA let-7c, known to negatively control K-Ras transcription. CONCLUSIONS: Our results suggest a novel cross-talk between Gal-3-mediated downregulation of let 7c microRNA (which in turn negatively regulates K-Ras transcription and elucidates the association among Gal-3 let-7c and K-Ras transcription/translation, cellular compartmentalization and activity.

Non-Smad signaling pathways.

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Mu, Yabing; Gudey, Shyam Kumar; Landström, Maréne

2012-01-01

Transforming growth factor-beta (TGFβ) is a key regulator of cell fate during embryogenesis and has also emerged as a potent driver of the epithelial-mesenchymal transition during tumor progression. TGFβ signals are transduced by transmembrane type I and type II serine/threonine kinase receptors (TβRI and TβRII, respectively). The activated TβR complex phosphorylates Smad2 and Smad3, converting them into transcriptional regulators that complex with Smad4. TGFβ also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Ubiquitin ligase tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) and TGFβ-associated kinase 1 (TAK1) have recently been shown to be crucial for the activation of the p38 and JNK MAPK pathways. Other TGFβ-induced non-Smad signaling pathways include the phosphoinositide 3-kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-MAPK pathway. Signals induced by TGFβ are tightly regulated and specified by post-translational modifications of the signaling components, since they dictate the subcellular localization, activity, and duration of the signal. In this review, we discuss recent findings in the field of TGFβ-induced responses by non-Smad signaling pathways.

Hydrogen Peroxide Toxicity Induces Ras Signaling in Human Neuroblastoma SH-SY5Y Cultured Cells

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Jirapa Chetsawang

2010-01-01

Full Text Available It has been reported that overproduction of reactive oxygen species occurs after brain injury and mediates neuronal cells degeneration. In the present study, we examined the role of Ras signaling on hydrogen peroxide-induced neuronal cells degeneration in dopaminergic neuroblastoma SH-SY5Y cells. Hydrogen peroxide significantly reduced cell viability in SH-SY5Y cultured cells. An inhibitor of the enzyme that catalyzes the farnesylation of Ras proteins, FTI-277, and a competitive inhibitor of GTP-binding proteins, GDP-beta-S significantly decreased hydrogen peroxide-induced reduction in cell viability in SH-SY5Y cultured cells. The results of this study might indicate that a Ras-dependent signaling pathway plays a role in hydrogen peroxide-induced toxicity in neuronal cells.

Induction of Non-Apoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

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Bhanot, Haymanti; Young, Ashley M.; Overmeyer, Jean H.; Maltese, William A.

2010-01-01

Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating-protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or co-expression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by shRNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1, combined with reciprocal inactivation of Arf6. The latter appears to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for induction of methuosis in cancers that are resistant to apoptotic cell death. PMID:20713492

Transformation by Oncogenic Ras Expands the Early Genomic Response to Transforming Growth Factor β in Intestinal Epithelial Cells

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Carl E. Allen

2008-10-01

Full Text Available A substantial body of evidence implicates TGFβ as a tumor promoter in epithelial cells that have become resistant to its tumor suppressor activity. To better understand early, genome-wide TGFβ responses in cells resistant to growth inhibition by TGFβ, we used microarray analysis in a well-defined cell culture system of sensitive and resistant intestinal epithelial cells. TGFβ-regulated gene expression in TGFβ-growth-sensitive, nontransformed rat intestinal epithelial cells (RIE-1 was compared to expression in TGFβ-growth-resistant RIE cells stably transformed by oncogenic Ras(12V. Treatment of RIE-1 cells with 2 ng/ml TGFβ1 for 1 hour increased the expression of eight gene sequences by 2.6-fold or more, whereas eight were down regulated 2.6-fold. In RIE-Ras(12V cells, 42 gene sequences were upregulated and only 3 were down-regulated. Comparison of RIE and RIE-Ras(12V identified 37 gene sequences as unique, Ras-dependent genomic targets of TGFβ1. TGFβ-regulation of connective tissue growth factor and vascular endothelial growth factor, two genes up-regulated in RIE-Ras cells and previously implicated in tumor promotion, was independently confirmed and further characterized by Northern analysis. Our data indicate that overexpression of oncogenic Ras in intestinal epithelial cells confers a significantly expanded repertoire of robust, early transcriptional responses to TGFβ via signaling pathways yet to be fully elucidated but including the canonical Raf-1/MAPK/Erk pathway. Loss of sensitivity to growth inhibition by TGFβ does not abrogate TGFβ signaling and actually expands the early transcriptional response to TGFβ1. Expression of some of these genes may confer to Ras-transformed cells characteristics favorable for tumor promotion.

Lymphatic endothelial S1P promotes mitochondrial function and survival in naive T cells.

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Mendoza, Alejandra; Fang, Victoria; Chen, Cynthia; Serasinghe, Madhavika; Verma, Akanksha; Muller, James; Chaluvadi, V Sai; Dustin, Michael L; Hla, Timothy; Elemento, Olivier; Chipuk, Jerry E; Schwab, Susan R

2017-06-01

Effective adaptive immune responses require a large repertoire of naive T cells that migrate throughout the body, rapidly identifying almost any foreign peptide. Because the production of T cells declines with age, naive T cells must be long-lived. However, it remains unclear how naive T cells survive for years while constantly travelling. The chemoattractant sphingosine 1-phosphate (S1P) guides T cell circulation among secondary lymphoid organs, including spleen, lymph nodes and Peyer's patches, where T cells search for antigens. The concentration of S1P is higher in circulatory fluids than in lymphoid organs, and the S1P 1 receptor (S1P 1 R) directs the exit of T cells from the spleen into blood, and from lymph nodes and Peyer's patches into lymph. Here we show that S1P is essential not only for the circulation of naive T cells, but also for their survival. Using transgenic mouse models, we demonstrate that lymphatic endothelial cells support the survival of T cells by secreting S1P via the transporter SPNS2, that this S1P signals through S1P 1 R on T cells, and that the requirement for S1P 1 R is independent of the established role of the receptor in guiding exit from lymph nodes. S1P signalling maintains the mitochondrial content of naive T cells, providing cells with the energy to continue their constant migration. The S1P signalling pathway is being targeted therapeutically to inhibit autoreactive T cell trafficking, and these findings suggest that it may be possible simultaneously to target autoreactive or malignant cell survival.

RAS Initiative - Community Outreach

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Through community and technical collaborations, workshops and symposia, and the distribution of reference reagents, the RAS Initiative seeks to increase the sharing of knowledge and resources essential to defeating cancers caused by mutant RAS genes.

Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest

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Dina Dikovskaya

2015-09-01

Full Text Available Oncogene-induced senescence (OIS is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

International Nuclear Information System (INIS)

Kolokoltsova, Olga A.; Domina, Aaron M.; Kolokoltsov, Andrey A.; Davey, Robert A.; Weaver, Scott C.; Watowich, Stanley J.

2008-01-01

Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection

Carprofen induction of p75NTR-dependent apoptosis via the p38 mitogen-activated protein kinase pathway in prostate cancer cells.

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Khwaja, Fatima S; Quann, Emily J; Pattabiraman, Nagarajan; Wynne, Shehla; Djakiew, Daniel

2008-11-01

The p75 neurotrophin receptor (p75(NTR)) functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75(NTR) expression in several prostate cancer cell lines leading to p75(NTR)-mediated decreased survival. Using the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico database of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75(NTR) levels and inhibition of cell survival. Prostate (PC-3 and DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75(NTR)-associated loss of survival than breast (MCF-7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant-negative form of p75(NTR) before carprofen treatment partially rescued cell survival, showing a cause-and-effect relationship between carprofen induction of p75(NTR) levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF-7 and 3T3 cells. Furthermore, small interfering RNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75(NTR) by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 min. Expression of a dominant-negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75(NTR) protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75(NTR)-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells.

20(S-ginsenoside Rg3 promotes senescence and apoptosis in gallbladder cancer cells via the p53 pathway

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Zhang F

2015-08-01

Full Text Available Fei Zhang,* Maolan Li,* Xiangsong Wu,* Yunping Hu, Yang Cao, Xu’an Wang, Shanshan Xiang, Huaifeng Li, Lin Jiang, Zhujun Tan, Wei Lu, Hao Weng, Yijun Shu, Wei Gong, Xuefeng Wang, Yong Zhang, Weibin Shi, Ping Dong,# Jun Gu,# Yingbin Liu#Department of General Surgery and Laboratory of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Institute of Biliary Tract Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work #These authors jointly directed this work Abstract: Gallbladder cancer (GBC, the most frequent malignancy of the biliary tract, is associated with high mortality and extremely poor prognosis. 20(S-ginsenoside Rg3 (20(S-Rg3 is a steroidal saponin with high pharmacological activity. However, the anticancer effect of 20(S-Rg3 in human GBC has not yet been determined. In this study, we primarily found that 20(S-Rg3 exposure suppressed the survival of both NOZ and GBC-SD cell lines in a concentration-dependent manner. Moreover, induction of cellular senescence and G0/G1 arrest by 20(S-Rg3 were accompanied by a large accumulation of p53 and p21 as a result of murine double minute 2 (MDM2 inhibition. 20(S-Rg3 also caused a remarkable increase in apoptosis via the activation of the mitochondrial-mediated intrinsic caspase pathway. Furthermore, intraperitoneal injection of 20(S-Rg3 (20 or 40 mg/kg for 3 weeks markedly inhibited the growth of xenografts in nude mice. Our results demonstrated that 20(S-Rg3 potently inhibited growth and survival of GBC cells both in vitro and in vivo. 20(S-Rg3 attenuated GBC growth probably via activation of the p53 pathway, and subsequent induction of cellular senescence and mitochondrial-dependent apoptosis. Therefore, 20(S-Rg3 may be a potential chemotherapeutic agent for GBC therapy.Keywords: gallbladder cancer, 20(S-ginsenoside Rg3, senescence, apoptosis, p53 pathway

MicroRNA-21-Mediated Inhibition of Mast Cell Degranulation Involved in the Protective Effect of Berberine on 2,4-Dinitrofluorobenzene-Induced Allergic Contact Dermatitis in Rats via p38 Pathway.

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Li, Weihua; Liu, Fanxiu; Wang, Jun; Long, Man; Wang, Zhigang

2018-03-01

The study aimed to investigate the effect of berberine on allergic contact dermatitis (ACD) in rats and explore its underlying mechanisms. Firstly, ACD model was established by sensitizing and challenging with 2,4-dinitrofluorobenzene (DNFB) topically, and the rats were treated with berberine. Ear swelling was assessed, and cytokine, IgE, and histamine productions were measured. The ear biopsies were obtained for histology analysis. Additionally, rat peritoneal mast cells (RPMCs) were isolated for detection of microRNA-21 (miR-21) expression, mitogen-activated protein kinase (MAPK) signaling, and MC degranulation. Lastly, RPMCs were transfected with miR-21 mimic or miR-21 inhibitor to investigate the relationship between miR-21 and p38 pathway in MC. Our results showed that berberine significantly attenuated ear swelling in DNFB-induced ACD (ACD vs high dose of berberine 0.48 ± 0.03 vs. 0.33 ± 0.03 mm, P < 0.01), inhibited inflammatory cell infiltration (86 ± 5.16 vs. 58 ± 4.32 cells/mm 2 , P < 0.01), reduced MC recruitment (61 ± 4.07 vs. 39 ± 3.42 mast cells/mm 2 , P < 0.01), as well as decreased inflammatory cytokine, IgE, and histamine productions (all P < 0.05). Berberine treatment inhibited miR-21 expression, suppressed β-hexosaminidase and histamine release, and prevented p38 phosphorylation (all P < 0.05), which was abrogated by pretreatment with miR-21 overexpression. These findings indicate that miR-21-mediated inhibition of MC degranulation is involved in the anti-ACD effect of berberine via inhibiting p38 pathway, which provide a new insight into the immunopharmacological role of berberine and suggest its potential application for the treatment of allergic inflammation, such as ACD.

Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.

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Kai Zhao

Full Text Available The patients diagnosed with melanoma have a bad prognosis for early regional invasion and distant metastases. Wogonin (5,7-dihydroxy-8-methoxyflavone is one of the active components of flavonoids that extracts from Scutellariae radix. Several previous studies reported that wogonin possesses antitumor effect against leukemia, gastrointestinal cancer and breast cancer. In this study, we used melanoma cell B16-F10 to further investigate the anti-invasive and anti-migratory activity of wogonin. Our date showed that wogonin caused suppression of cell migration, adhesion, invasion and actin remodeling by inhibiting the expression of matrix metalloproteinase-2 and Rac1 in vitro. Wogonin also reduced the number of the tumor nodules on the whole surface of the lung in vivo. Furthermore, the examination of mechanism revealed that wogonin inhibited Extracellular Regulated protein Kinases and Protein Kinase B pathways, which are both medicated by Ras. Insulin-like growth factor-1-induced or tumor necrosis factor-α-induced invasion was also inhibited by wogonin. Therefore, the inhibitory mechanism of melanoma cell invasion by wogonin might be elucidated.

K-ras2 Activation and Genome Instability Increase Proliferation and Size of FAP Adenomas

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Anna Rapallo

1999-01-01

Full Text Available The possible role of K‐ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%, low grade dysplasia (63%, and were preferentially located in the right colon (60%. Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K‐ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO hybridization, while flow cytometry (FCM was used for evaluating degree of DNA ploidy and S‐phase fraction. Overall, incidences of K‐ras2 mutations, DNA aneuploidy and high S‐phase values (>7.2% were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K‐ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K‐ras2 and size, DNA ploidy and size and K‐ras2 and S‐phase (p. In particular, among the wild type K‐ras2 adenomas, high S‐phase values were detected in 8% of the cases versus 57% among the K‐ras2 mutated adenomas (p=0.0005. The present series of FAP adenomas indicates that K‐ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.

Ferulago angulata activates intrinsic pathway of apoptosis in MCF-7 cells associated with G1 cell cycle arrest via involvement of p21/p27

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Karimian H

2014-09-01

Full Text Available Hamed Karimian,1 Soheil Zorofchian Moghadamtousi,2 Mehran Fadaeinasab,3 Shahram Golbabapour,2 Mahboubeh Razavi,1 Maryam Hajrezaie,2 Aditya Arya,1 Mahmood Ameen Abdulla,4 Syam Mohan,5 Hapipah Mohd Ali,2 Mohamad Ibrahim Noordin1 1Department of Pharmacy, Faculty of Medicine, 2Institute of Biological Sciences, Faculty of Science, 3Department of Chemistry, 4Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 5Medical Research Centre, Jazan University, Jazan, Saudi Arabia Abstract: Ferulago angulata is a medicinal plant that is traditionally known for its ­anti-inflammatory and antiulcer properties. The present study was aimed to evaluate its anticancer activity and the possible mechanism of action using MCF-7 as an in vitro model. F. angulata leaf extracts were prepared using solvents in the order of increasing polarity. As determined by MTT assay, F. angulata leaves hexane extract (FALHE revealed the strongest cytotoxicity against MCF-7 cells with the half maximal inhibitory concentration (IC50 value of 5.3±0.82 µg/mL. The acute toxicity study of FALHE provided evidence of the safety of the plant extract. Microscopic and flow cytometric analysis using annexin-V probe showed an induction of apoptosis in MCF-7 by FALHE. Treatment of MCF-7 cells with FALHE encouraged the intrinsic pathway of apoptosis, with cell death transducing signals that reduced the mitochondrial membrane potential with cytochrome c release from mitochondria to cytosol. The released cytochrome c triggered the activation of caspase-9. Meanwhile, the overexpression of caspase-8 suggested the involvement of an extrinsic pathway in the induced apoptosis at the late stage of treatment. Moreover, flow cytometric analysis showed that FALHE treatment significantly arrested MCF-7 cells in the G1 phase, which was associated with upregulation of p21 and p27 assessed by quantitative polymerase chain reaction. Immunofluorescence

Regulation and Selectivity of Exchange Factors for G-proteins of the Ras-family

NARCIS (Netherlands)

Popovic, M.

2013-01-01

Small G-proteins are important regulators of the cellular signaling pathways. Among them, members of the Ras family of small G-proteins regulate processes such as cell differentiation, growth, migration, transport and adhesion, and their deregulation may lead to various diseases. Small G-proteins

Msn2p/Msn4p act as a key transcriptional activator of yeast cytoplasmic thiol peroxidase II.

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Hong, Seung-Keun; Cha, Mee-Kyung; Choi, Yong-Soo; Kim, Won-Cheol; Kim, Il-Han

2002-04-05

We observed that the transcription of Saccharomyces cerevisiae cytoplasmic thiol peroxidase type II (cTPx II) (YDR453C) is regulated in response to various stresses (e.g. oxidative stress, carbon starvation, and heat-shock). It has been suggested that both transcription-activating proteins, Yap1p and Skn7p, regulate the transcription of cTPx II upon exposure to oxidative stress. However, a dramatic loss of transcriptional response to various stresses in yeast mutant strains lacking both Msn2p and Msn4p suggests that the transcription factors act as a principal transcriptional activator. In addition to two Yap1p response elements (YREs), TTACTAA and TTAGTAA, the presence of two stress response elements (STREs) (CCCCT) in the upstream sequence of cTPx II also suggests that Msn2p/Msn4p could control stress-induced expression of cTPx II. Analysis of the transcriptional activity of site-directed mutagenesis of the putative STREs (STRE1 and STRE2) and YREs (TRE1 and YRE2) in terms of the activity of a lacZ reporter gene under control of the cTPx II promoter indicates that STRE2 acts as a principal binding element essential for transactivation of the cTPx II promoter. The transcriptional activity of the cTPx II promoter was exponentially increased after postdiauxic growth. The transcriptional activity of the cTPx II promoter is greatly increased by rapamycin. Deletion of Tor1, Tor2, Ras1, and Ras2 resulted in a considerable induction when compared with their parent strains, suggesting that the transcription of cTPx II is under negative control of the Ras/cAMP and target of rapamycin signaling pathways. Taken together, these results suggest that cTPx II is a target of Msn2p/Msn4p transcription factors under negative control of the Ras-protein kinase A and target of rapamycin signaling pathways. Furthermore, the accumulation of cTPx II upon exposure to oxidative stress and during the postdiauxic shift suggests an important antioxidant role in stationary phase yeast cells.

Effect of Primary Tumor Location on Second- or Later-line Treatment Outcomes in Patients With RAS Wild-type Metastatic Colorectal Cancer and All Treatment Lines in Patients With RAS Mutations in Four Randomized Panitumumab Studies.

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Boeckx, Nele; Koukakis, Reija; Op de Beeck, Ken; Rolfo, Christian; Van Camp, Guy; Siena, Salvatore; Tabernero, Josep; Douillard, Jean-Yves; André, Thierry; Peeters, Marc

2018-03-08

The primary tumor location has a prognostic impact in metastatic colorectal cancer (mCRC). We report the results from retrospective analyses assessing the effect of tumor location on prognosis and efficacy of second- and later-line panitumumab treatment in patients with RAS wild-type (WT) mCRC and on prognosis in all lines of treatment in patients with RAS mutant (MT) mCRC. RAS WT data (n = 483) from 2 randomized phase III panitumumab trials (ClinicalTrials.gov identifiers, NCT00339183 and NCT00113763) were analyzed for treatment outcomes stratified by tumor location. The second analysis assessed the effect of tumor location in RAS MT patients (n = 1205) from 4 panitumumab studies (ClinicalTrials.gov identifiers, NCT00364013, NCT00819780, NCT00339183, and NCT00113763). Primary tumors located in the cecum to transverse colon were coded as right-sided; those located from the splenic flexure to the rectum were coded as left-sided. Of all patients, the tumor location was ascertained for 83% to 88%; 71% to 77% of patients had left-sided tumors. RAS WT patients with right-sided tumors did worse for all efficacy parameters compared with those with left-sided tumors. The patients with left-sided tumors had better outcomes with panitumumab than with the comparator treatment. Because of the low patient numbers, no conclusions could be drawn for right-sided mCRC. The prognostic effect of tumor location on survival was unclear for RAS MT patients. These retrospective analyses have confirmed that RAS WT right-sided mCRC is associated with a poor prognosis, regardless of the treatment. RAS WT patients with left-sided tumors benefitted from the addition of panitumumab in second or later treatment lines. Further research is warranted to determine the optimum management of right-sided mCRC and RAS MT tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

A microRNA activity map of human mesenchymal tumors: connections to oncogenic pathways; an integrative transcriptomic study

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Fountzilas Elena

2012-07-01

Full Text Available Abstract Background MicroRNAs (miRNAs are nucleic acid regulators of many human mRNAs, and are associated with many tumorigenic processes. miRNA expression levels have been used in profiling studies, but some evidence suggests that expression levels do not fully capture miRNA regulatory activity. In this study we integrate multiple gene expression datasets to determine miRNA activity patterns associated with cancer phenotypes and oncogenic pathways in mesenchymal tumors – a very heterogeneous class of malignancies. Results Using a computational method, we identified differentially activated miRNAs between 77 normal tissue specimens and 135 sarcomas and we validated many of these findings with microarray interrogation of an independent, paraffin-based cohort of 18 tumors. We also showed that miRNA activity is imperfectly correlated with miRNA expression levels. Using next-generation miRNA sequencing we identified potential base sequence alterations which may explain differential activity. We then analyzed miRNA activity changes related to the RAS-pathway and found 21 miRNAs that switch from silenced to activated status in parallel with RAS activation. Importantly, nearly half of these 21 miRNAs were predicted to regulate integral parts of the miRNA processing machinery, and our gene expression analysis revealed significant reductions of these transcripts in RAS-active tumors. These results suggest an association between RAS signaling and miRNA processing in which miRNAs may attenuate their own biogenesis. Conclusions Our study represents the first gene expression-based investigation of miRNA regulatory activity in human sarcomas, and our findings indicate that miRNA activity patterns derived from integrated transcriptomic data are reproducible and biologically informative in cancer. We identified an association between RAS signaling and miRNA processing, and demonstrated sequence alterations as plausible causes for differential miRNA activity

MiR-23a-5p modulates mycobacterial survival and autophagy during mycobacterium tuberculosis infection through TLR2/MyD88/NF-κB pathway by targeting TLR2.

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Gu, Xing; Gao, Yan; Mu, De-Guang; Fu, En-Qing

2017-05-15

Autophagy plays a pivotal role in activating the antimicrobial host defense against Mycobacterium tuberculosis (M.tb.). The emerging roles of microRNAs (miRNAs) in regulating immune responses have attracted increasing attention in recent years. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the influence of miR-23a-5p on the activation of macrophage autophagy during M.tb. infection in bone marrow-derived macrophages (BMDMs) and murine RAW264.7 cells. Here, we demonstrated that M.tb.-infection of macrophages lead to markedly enhanced expression of miR-23a-5p in a time- and dose-dependent manner. Furthermore, forced expression of miR-23a-5p accelerated the survival rate of intracellular mycobacteria, while transfection with miR-23a-5p inhibitors attenuated mycobacterial survival. More importantly, overexpression of miR-23a-5p dramatically prevented M.tb.-induced activation of autophagy in macrophages, whereas inhibitors of miR-23a-5p remarkably accelerated M.tb.-induced autophagy. Mechanistically, miR-23a-5p is able to modulate TLR2/MyD88/NF-κB signaling activity by targeting TLR2 in RAW264.7 cells in response to M.tb.-infection. Collectively, these findings demonstrated that miR-23a-5p modulated the innate host defense by promoting mycobacteria survival and inhibiting the activation of autophagy against M.tb. through TLR2/MyD88/NF-κB pathway by targeting TLR2, which may provide a promising therapeutic target for tuberculosis. Copyright © 2017 Elsevier Inc. All rights reserved.

High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

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Chavan, Tanmay S.; Jang, Hyunbum; Khavrutskii, Lyuba; Abraham, Sherwin J.; Banerjee, Avik; Freed, Benjamin C.; Johannessen, Liv; Tarasov, Sergey G.; Gaponenko, Vadim; Nussinov, Ruth; Tarasova, Nadya I.

2015-01-01

Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible. PMID:26682817

Overexpression of miR‑21 promotes neural stem cell proliferation and neural differentiation via the Wnt/β‑catenin signaling pathway in vitro.

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Zhang, Wei-Min; Zhang, Zhi-Ren; Yang, Xi-Tao; Zhang, Yong-Gang; Gao, Yan-Sheng

2018-01-01

The primary aim of the present study was to examine the effects of microRNA‑21 (miR‑21) on the proliferation and differentiation of rat primary neural stem cells (NSCs) in vitro. miR‑21 was overexpressed in NSCs by transfection with a miR‑21 mimic. The effects of miR‑21 overexpression on NSC proliferation were revealed by Cell Counting kit 8 and 5‑ethynyl‑2'‑deoxyuridine incorporation assay, and miR‑21 overexpression was revealed to increase NSC proliferation. miR‑21 overexpression was confirmed using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). mRNA and protein expression levels of key molecules (β‑catenin, cyclin D1, p21 and miR‑21) in the Wnt/β‑catenin signaling pathway were studied by RT‑qPCR and western blot analysis. RT‑qPCR and western blot analyses revealed that miR‑21 overexpression increased β‑catenin and cyclin D1 expression, and decreased p21 expression. These results suggested that miR‑21‑induced increase in proliferation was mediated by activation of the Wnt/β‑catenin signaling pathway, since overexpression of miR‑21 increased β‑catenin and cyclin D1 expression and reduced p21 expression. Furthermore, inhibition of the Wnt/β‑catenin pathway with FH535 attenuated the influence of miR‑21 overexpression on NSC proliferation, indicating that the factors activated by miR‑21 overexpression were inhibited by FH535 treatment. Furthermore, overexpression of miR‑21 enhanced the differentiation of NSCs into neurons and inhibited their differentiation into astrocytes. The present study indicated that in primary rat NSCs, overexpression of miR‑21 may promote proliferation and differentiation into neurons via the Wnt/β‑catenin signaling pathway in vitro.

Implementing Toxicity Testing in the 21st Century (TT21C): Making safety decisions using toxicity pathways, and progress in a prototype risk assessment.

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Adeleye, Yeyejide; Andersen, Melvin; Clewell, Rebecca; Davies, Michael; Dent, Matthew; Edwards, Sue; Fowler, Paul; Malcomber, Sophie; Nicol, Beate; Scott, Andrew; Scott, Sharon; Sun, Bin; Westmoreland, Carl; White, Andrew; Zhang, Qiang; Carmichael, Paul L

2015-06-05

Risk assessment methodologies in toxicology have remained largely unchanged for decades. The default approach uses high dose animal studies, together with human exposure estimates, and conservative assessment (uncertainty) factors or linear extrapolations to determine whether a specific chemical exposure is 'safe' or 'unsafe'. Although some incremental changes have appeared over the years, results from all new approaches are still judged against this process of extrapolating high-dose effects in animals to low-dose exposures in humans. The US National Research Council blueprint for change, entitled Toxicity Testing in the 21st Century: A Vision and Strategy called for a transformation of toxicity testing from a system based on high-dose studies in laboratory animals to one founded primarily on in vitro methods that evaluate changes in normal cellular signalling pathways using human-relevant cells or tissues. More recently, this concept of pathways-based approaches to risk assessment has been expanded by the description of 'Adverse Outcome Pathways' (AOPs). The question, however, has been how to translate this AOP/TT21C vision into the practical tools that will be useful to those expected to make safety decisions. We have sought to provide a practical example of how the TT21C vision can be implemented to facilitate a safety assessment for a commercial chemical without the use of animal testing. To this end, the key elements of the TT21C vision have been broken down to a set of actions that can be brought together to achieve such a safety assessment. Such components of a pathways-based risk assessment have been widely discussed, however to-date, no worked examples of the entire risk assessment process exist. In order to begin to test the process, we have taken the approach of examining a prototype toxicity pathway (DNA damage responses mediated by the p53 network) and constructing a strategy for the development of a pathway based risk assessment for a specific

Mutations in APC, CTNNBI en K-ras genes and expression of hMLHI in sporadic colorectal carcinomas from the Netherlands Cohort Study

NARCIS (Netherlands)

Luchtenborg, M.; Weijenberg, M.P.; Wark, P.A.; Merdan Saritas, M.

2005-01-01

Background - The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of

Metastatic triple-negative breast cancer is dependent on SphKs/S1P signaling for growth and survival.

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Maiti, Aparna; Takabe, Kazuaki; Hait, Nitai C

2017-04-01

About 40,000 American women die from metastatic breast cancer each year despite advancements in treatment. Approximately, 15% of breast cancers are triple-negative for estrogen receptor, progesterone receptor, and HER2. Triple-negative cancer is characterized by more aggressive, harder to treat with conventional approaches and having a greater possibility of recurrence. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid signaling mediator has emerged as a key regulatory molecule in breast cancer progression. Therefore, we investigated whether cytosolic sphingosine kinase type 1 (SphK1) and nuclear sphingosine kinase type 2 (SphK2), the enzymes that make S1P are critical for growth and PI3K/AKT, ERK-MAP kinase mediated survival signaling of lung metastatic variant LM2-4 breast cancer cells, generated from the parental triple-negative MDA-MB-231 human breast cancer cell line. Similar with previous report, SphKs/S1P signaling is critical for the growth and survival of estrogen receptor positive MCF-7 human breast cancer cells, was used as our study control. MDA-MB-231 did not show a significant effect of SphKs/S1P signaling on AKT, ERK, and p38 pathways. In contrast, LM2-4 cells that gained lung metastatic phenotype from primary MDA-MB-231 cells show a significant effect of SphKs/S1P signaling requirement on cell growth, survival, and cell motility. PF-543, a selective potent inhibitor of SphK1, attenuated epidermal growth factor (EGF)-mediated cell growth and survival signaling through inhibition of AKT, ERK, and p38 MAP kinase pathways mainly in LM2-4 cells but not in parental MDA-MB-231 human breast cancer cells. Moreover, K-145, a selective inhibitor of SphK2, markedly attenuated EGF-mediated cell growth and survival of LM2-4 cells. We believe this study highlights the importance of SphKs/S1P signaling in metastatic triple-negative breast cancers and targeted therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulatory module involving FGF13, miR-504, and p53 regulates ribosomal biogenesis and supports cancer cell survival

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Bublik, Débora R.; Bursać, Slađana; Sheffer, Michal; Oršolić, Ines; Shalit, Tali; Tarcic, Ohad; Kotler, Eran; Mouhadeb, Odelia; Hoffman, Yonit; Fuchs, Gilad; Levin, Yishai; Volarević, Siniša; Oren, Moshe

2017-01-01

The microRNA miR-504 targets TP53 mRNA encoding the p53 tumor suppressor. miR-504 resides within the fibroblast growth factor 13 (FGF13) gene, which is overexpressed in various cancers. We report that the FGF13 locus, comprising FGF13 and miR-504, is transcriptionally repressed by p53, defining an additional negative feedback loop in the p53 network. Furthermore, we show that FGF13 1A is a nucleolar protein that represses ribosomal RNA transcription and attenuates protein synthesis. Importantly, in cancer cells expressing high levels of FGF13, the depletion of FGF13 elicits increased proteostasis stress, associated with the accumulation of reactive oxygen species and apoptosis. Notably, stepwise neoplastic transformation is accompanied by a gradual increase in FGF13 expression and increased dependence on FGF13 for survival (“nononcogene addiction”). Moreover, FGF13 overexpression enables cells to cope more effectively with the stress elicited by oncogenic Ras protein. We propose that, in cells in which activated oncogenes drive excessive protein synthesis, FGF13 may favor survival by maintaining translation rates at a level compatible with the protein quality-control capacity of the cell. Thus, FGF13 may serve as an enabler, allowing cancer cells to evade proteostasis stress triggered by oncogene activation. PMID:27994142

P120-GAP associated with syndecan-2 to function as an active switch signal for Src upon transformation with oncogenic ras

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Huang, J.-W.; Chen, C.-L.; Chuang, N.-N.

2005-01-01

BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q 61 K)] of shrimp Penaeus japonicus were applied to reveal a complex of p120-GAP/syndecan-2 being highly expressed upon transformation. Of interest, most of the p120-GAP/syndecan-2 complex was localized at caveolae, a membrane microdomain enriched with caveolin-1. To confirm the molecular interaction between syndecan-2 and p120-GAP, we further purified p120-GAP protein from mouse brains by using an affinity column of HiTrap-RACK1 and expressed mouse RACK1-encoded fusion protein and mouse syndecan-2-encoded fusion protein in bacteria. We report molecular affinities exist between p120-GAP and RACK1, syndecan-2 and RACK1 as well as p120-GAP and syndecan-2. The selective affinity between p120-GAP and syndecan-2 was found to be sufficient to detach RACK1. The p120-GAP/syndecan-2 complex was demonstrated to keep Src tyrosine kinase in an activated form. On the other hand, the syndecan-2/RACK1 complex was found to have Src in an inactivated form. These data indicate that the p120-GAP/syndecan-2 complex at caveolae could provide a docking site for Src to transmit tyrosine signaling, implying that syndecan-2/p120-GAP functions as a tumor promoter upon transformation with oncogenic ras of shrimp P. japonicus

Ras proteins have multiple functions in vegetative cells of Dictyostelium.

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Bolourani, Parvin; Spiegelman, George; Weeks, Gerald

2010-11-01

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

Urine RAS components in mice and people with type 1 diabetes and chronic kidney disease.